rna polymerase and associated factors, part b

EMERSON 24, Regulatory Biol- ogy Laboratory, The Salk Institute for Bio- logical Studies, La Jolla, California 92037 GARY FELSENFELI 19, Laboratory of Mo- lecular Biology, National In

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P r e f a c e One cannot fully understand the biology of a cell without understanding the central role that gene expression and its regulation play RNA polymerase was discovered in eukaryotes in 1959 and in prokaryotes in 1960, and the subject of transcription regulation was reported in the early 1960s Although many breakthrough experiments were performed in the 1970s, there has been, unquestionably, an explosion in our knowledge in the field since the 1980s, thanks to the rapid development and use of powerful genetic, biochemical, and physical techniques As a result, many plausible, sometimes unexpected, ideas have been generated More encouragingly, some of the ideas have been accepted

Volumes 273 and 274 of Methods in Enzymology cover, for the first

time, methods and other analytical approaches for the study of transcription and its regulation in prokaryotes and eukaryotes The chapters in these two volumes describe steps of transcription; component machinery and their specificity; purification, assays, and properties of RNA polymerases and their intrinsic and extrinsic (including regulatory) factors that guide transcription initiation, elongation, and termination; and the assembly of RNA polymerase holoenzymes and many regulatory protein-protein and nucleoprotein complexes, including chromatins A few chapters dealing with specialized techniques analyzing transcriptional regulation are also included

These volumes will help further exploration of how transcription con- trols cellular adaptation, development, and differentiation We underscore the importance of DNA-protein interactions in studying transcription and its regulation, a subject covered in Volume 208 of this series

SANKAR ADHYA

xvii

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C o n t r i b u t o r s to V o l u m e 2 7 4

Article numbers are in parentheses following the names of contributors

Affiliations listed are current

CHRISTOPHER C ADAMS (22), Department

of Biochemistry and Molecular Biology,

Pennsylvania State University, University

Park, Pennsylvania 16802

AMANDA S ALTIERI (30), Macromolecular

NMR Section, ABL-Basic Research Pro-

gram, NCI-Frederick Cancer Research and

Development Center, Frederick, Maryland

21702

TEIJIRO ASO (33), Program in Molecular and

Cell Biology, Oklahoma Medical Research

Foundation, Oklahoma City, Oklahoma

73104

SAILEN BARIK (29), Department of Biochem-

istry and Molecular Biology, University of

South Alabama College of Medicine, Mo-

bile, Alabama 36688

M1CHELLE CRAIG BARTON (24), Department

of Molecular Genetics, University of Cincin-

nati, Cincinnati, Ohio 45267

CONSTANZE BONIFER (18), Institut ftir Biolo-

gie lII, Albert-Ludwigs-Universitdt Frei-

burg, D-79104 Freiburg, Germany

UWE BORGMEYER (18), Center for Molecular

Neurobiology II, University of Hamburg,

D-20251 Hamburg, Germany

SERGE1 BORUKHOV (25, 26), Department of

Microbiology and Immunology, State Uni-

versity of New York, Health Science Center

at Brooklyn, Brooklyn, New York 11203

MICHAEL BRENOWITZ (36), Department of

Biochemistry, Albert Einstein College of

Medicine, Bronx, New York 10461

RICHARD R BURGESS (39), McArdle Labora-

tory for Cancer Research, University of

Wisconsin-Madison, Madison, Wisconsin

53706

SANDEEP BURMA (3), Eukaryotic Gene Ex-

pression Laboratory, National Institute of

Immunology, New Delhi-llO067, India

R ANDREW BYRD (30), Macromolecular NMR Section, ABL-Basic Research Pro- gram, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland

21702

MICHAEL CAREY (11), Department of Biologi- cal Chemistry, UCLA School of Medicine, Los Angeles, California 90095

CATHLEEN L CHAN (27), Department of Sto- matology, University of California, San Francisco, California 94025

DIPANKAR CHATTERJI (35), Center for Cellu- lar and Molecular Biology, Hyderabad-500

007 (A.P.), India

SAMIT CHATTOPADHYAY (30), Center for Can- cer Research, Department of Biology, Mas- sachusetts Institute of Technology, Cam- bridge, Massachusetts 02138

TIANHUAI CHI (11), Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California 90095

CHENG-MING CHIANG (6), Department of Biochemistry, University of Illinob at Ur- bana-Champaign, Urbana, Illinois 61801 M1EYOUNG CHOI (2), Committee on Develop- mental Biology, University of Chicago, Chi- cago, Illinois 60637

HYON E Cnov (1), Department of Molecular Biology, Odense University, DK-5230 Odense M, Denmark 20892-4255

DAVID J CLARK (19), Laboratory of Cellular and Developmental Biology, National Insti- tute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

JOAN WELIKY CONAWAY (33), Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104

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xii CONTRIBUTORS TO VOLUME 274

RONALD C CONAWAY (33), Program in Mo-

lecular and Cell Biology, Oklahoma Medi-

cal Research Foundation, Oklahoma City,

Oklahoma 73104

NINA COSTANTINO (30), Laboratory of Chro-

mosome Biology and ABL-Basic Research

Program, NC1-Frederick Cancer Research

and Development Center, Frederick, Mary-

land 21702

JACQUES COT 15 (22), Department of Biochem-

istry and Molecular Biology, Pennsylvania

State University, University Park, Pennsyl-

vania 16802

DONALD COURT (30), Laboratory of Chromo-

some Biology and ABL-Basic Research

Program, NCl-Frederick Cancer Research

and Development Center, Frederick, Mary-

land 21702

ROBIN CROSSLEY (30), Department of Micro-

biology, University of Connecticut School

of Medicine, Farmington, Connecticut

O6O30

XING DAI (2), Department of Biochemistry

and Molecular Biology, University of Chi-

cago, Chicago, Illinois 60637

AsIs DAS (29, 30), Department of Microbiol-

ogy, University of Connecticut School of

Medicine, Farmington, Connecticut 06030

JOSEPH DEVITo (30), Laboratory of Myco-

bacteria, Center for Biologics Evaluation

and Research, FDA, Bethesda, Maryland

20892

RONNY DRAPKIN (7), Department of Bio-

chemistry, Howard Hughes Medical Insti-

tute, Robert Wood Johnson Medical School,

University of Medicine and Dentistry of

New Jersey, Piscataway, New Jersey 08854

RICHARD H EBRIGHT (37), Department of

Chemistry and Waksman Institute, Rutgers

University, New Brunswick, New Jersey

O8855

ALED M EDWARDS (32), Cancer Research

Group, Institute for Molecular Biology and

Biotechnology, McMaster University, Ham-

ilton, Ontario L8N 3Z5, Canada

BEVERLY M EMERSON (24), Regulatory Biol-

ogy Laboratory, The Salk Institute for Bio-

logical Studies, La Jolla, California 92037

GARY FELSENFELI) (19), Laboratory of Mo- lecular Biology, National Institute of Diabe- tes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

MICHAEL FRITSCH (9), Laboratory of Bio- chemistry, National Cancer Institute, Na- tional Institutes of Health, Bethesda, Mary- land 20892

Hui GE (6), Laboratory of Molecular Embry- ology, National Institute of Child Health and Human Development, National Insti- tutes of Health, Bethesda, Maryland 20892

JEFFREY S GERBER (5), Laboratory of Molec- ular and Cellular Biology, National Institute

of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

BALARAM GHOSH (29, 30), Center for Bio- chemical Technology, Council of Scientific and Industrial Research, Delhi 110007, India

M ALEXANDRA GLUCKSMANN-KuIs (2), De- partment of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637

ALEX GOLDFARB (25, 26), Public Health Re- search Institute, New York, New York lO016

NORA GOOSEN (4), Laboratory of Molecular Genetics', Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories,

2300 RA Leiden, The Netherlands

VIJAYA GOPAL (35), Center for Cellular and Molecular Biology, Hyderabad-500 007 (A.P.), India

JACK GREENBLATF (10), Banting and Best De- partment of Medical Research, Department

of Biochemistry, and Department of Molec- ular and Medical Genetics, University of To- ronto, Toronto, Ontario M5G 1L6, Canada

SAMAN HAB1B (3), Eukaryotic Gene Expres- sion Laboratory, National Institute of Im- munology, New Delhi-llO067, India

JONATHAN HAM (14), Eisai London Research Laboratories, University College London, London WC1E 6BT, United Kingdom

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CONTRIBUTORS TO VOLUME 2 7 4 x i i i MICHELLE M HANNA (31), Departments of

Chemistry and Biochemistry, University of

Oklahoma, Norman, Oklahoma 73109

8EYED E HASNAIN (3), Eukaryotic Gene Ex-

pression Laboratory, National Institute of

Immunology, New Delhi-llO067, India

TOMASZ HEYDUK (37), Department of Bio-

chemistry and Molecular Biology, St Louis

University School of Medicine, St Louis,

Missouri 63104

DEBORAH M HINTON (5), Laboratory of Mo-

lecular and Cellular Biology, National Insti-

tute of Diabetes and Digestive and Kidney

Diseases, National Institutes of Health,

Bethesda, Maryland 20892

H CHRISTOPH HOEFER (18), Institutfar Biolo-

gie llI, Albert-Ludwigs-Universiti~t Frei-

burg, D-79104 Freiburg, Germany

RODER1CK HORI (1 0 , Department of Biologi-

cal Chernistry, UCLA School of Medicine,

Los Angeles, California 90095

JINZHAO HOU (8), Arris Pharmaceutical Cor-

poration, South San Francisco, California

94080

MARK HSIEH (36), Department of Biochemis-

try, Albert Einstein College of Medicine,

Bronx, New York 10461

MA'VFHIAS C HUBER (18), Institutfiir Biologie

Ill, Albert-Ludwigs-Universitiit Freiburg,

D-79104 Freiburg, Germany

C JAMES INOLES (10), Banting and Best De-

partment of Medical Research, Department

of Biochemistry, and Department of Molec-

ular and Medical Genetics, University of

Toronto, Toronto, Ontario M5G 1L6,

Canada

ANJALI JA1N (3), Eukaryotic Gene Expression

Laboratory, National Institute of Immunol-

ogy, New Delhi-llO067, India

PAUL JEDLICKA (9), Laboratory of Biochemis-

try, National Cancer Institute, National

Institutes of Health, Bethesda, Maryland

20892

LI-JUNG JUAN (22), lntercollege Program in

Genetics, Pennsylvania State University,

University Park, Pennsylvania 16802

CAROLINE M KANE (32), Department of Mo- lecular and Cell Biology, University of Cali- fornia, Berkeley, California 94720

MIKHAIL KASHLEV (26), Public Health Re- search Institute, New York, New York 10016

MICHAEL P KLADDE (17), Department of Bio- chemistry and Molecular Biology, Pennsyl- vania State University, University Park, Pennsylvania 16802

RICHARD D KLAUSNER (33), Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

NATALIA KOMISSAROVA (26), Public Health Research Institute, New York, New York

10016

JOSEPH S KRAKOW (38), Department of Bio- logical Sciences, Hunter College of the City University of New York, New York, New York 10021

ROBERT LANDICK (27), Departments of Biol- ogy and Biochemistry and Molecular Bio- physics, Washington University, St Louis, Missouri 63130

W MARSTON LINEHAN (33), Urologic Oncol- ogy Section, Surgery Branch, National Can- cer Institute, National Institutes of Health, Bethesda, Maryland 20892

JIANYING LuO (38), Surgical Laboratory, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115

YUEX1NG MA (37), Department of Chemistry and Waksman Institute, Rutgers University, New Brunswick, New Jersey 08855

EDIO MALDONADO (7), Department of Bio- chemistry, Howard Hughes Medical Insti- tute, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854

TOM MANIAT1S (13), Department of Molecular and Cell Biology, Harvard University, Cam- bridge, Massachusetts 02138

ROSLYN MARCH-AMEGADZIE (5), Laboratory

of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kid- ney Diseases, National Institutes of Health, Bethesda, Maryland 20892

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x i v CONTRIBUTORS TO VOLUME 274

ERNEST MARTINEZ (6), Laboratory of Bio-

chemistry and Molecular Biology, The

Rockefeller University, New York, New

York 10021

MARIE MAZZULLA (30), Macromolecular

NMR Section, ABL-Basic Research Pro-

gram, NCI-Frederick Cancer Research and

Development Center, Frederick, Maryland

21702

STEVEN L McKNI~HT (8), Tularik Incorpo-

rated, South San Francisco, California

94080

JAIME OARCIA MENA (30), Department of

Microbiology, University of Connecticut

School of Medicine, Farmington, Connecti-

cut 06030

ALITA MILLER (2), Department of Biochemis-

try and Molecular Biology, University of

Chicago, Chicago, Illinois 60637

GAKU MIZUGUCHI (9), Laboratory of Bio-

chemistry, National Cancer Institute, Na-

tional Institutes of Health, Bethesda, Mary-

land 20892

TAKESHI MIZUNO (21), Laboratory of Molec-

ular Microbiology, School of Agriculture,

Nagoya University, Nagoya 464, Japan

BIPASHA MUKHEILIEE (3), Eukaryotic Gene

Expression Laboratory, National Institute

of Immunology, New Delhi-llO067, India

WILLIAM NOWATZKE (28), Department of

Chemistry, Indiana University, Blooming-

ton, Indiana 47405

EVGENY NUDLER (26), Public Health Re-

search Institute, New York, New York 10016

LAURA P O'NEILL (15), Chromatin and Gene

Expression Group, Anatomy Department,

University of Birmingham Medical School,

Edgbaston, Birmingham B15 2TT, United

Kingdom

ANDRAS OROSZ (9), Laboratory of Biochem-

istry, National Cancer Institute, National

20892

THOMAS A OWEN-HUGHES (22), Department

of Biochemistry and Molecular Biology,

Pennsylvania State University, University

Park, Pennsylvania 16802

MAHADEB PAL (30), Department of Microbi- ology, University of Connecticut School of Medicine, Farmington, Connecticut 06030

ARNIM PAUSE (33), Cell Biology and Metabo- lism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 2O892

SUNG PYo (11), Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California 90095

WILLIAM REES (30), Howard Hughes Medical Institute, National Jewish Center for Immu- nology and Respiratory Medicine, Denver, Colorado 80206

DANNY REINBERG (7), Department of Bio- chemistry, Howard Hughes Medical Insti- tute, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854

JOHN P RICHARDSON (28), Department of Chemistry, Indiana University, Blooming- ton, Indiana 47405

LisLoaT- RICHARDSON (28), Department of Chemistry, lndiana University, Blooming- ton, Indiana 47405

ROBERT G ROEDER (6), Laboratory of Bio- chemistry and Molecular Biology, The Rockefeller University, New York, New York 10021

LUCIA B ROTHMAN-DENES (2), Department

of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois

60637

HARALD SAUERESSIG (18), Molecular Neuro- biology Laboratory, The Salk Institute, La Jolla, California 92037

THOMAS D SCHNEIDER (34), Laboratory of Mathematical Biology, Frederick Cancer Research and Development Center, Na- tional Cancer Institute, Frederick, Mary- land 21702

KONSTANTIN SEVERINOV (26), Public Health Research Institute, New York, New York

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CONTRIBUTORS TO VOLUME 274 x v MRIDULA SHARMA (5), Laboratory of Molec-

ular and Cellular Biology, National Institute

of Diabetes and Digestive and Kidney

Diseases, National Institutes of Health,

Bethesda, Maryland 20892

ROBERT T SIMPSON (17), Department of Bio-

chemistry and Molecular Biology, Pennsyl-

vania State University, University Park,

Pennsylvania 16802

ALBRECHT E SIPPEL (18), Institutfar Biologie

III, Albert-Ludwigs-Universitiit Freiburg,

GERTRUD STEGER (14), Institut far Virologie,

Universitiit zu KOln, 50935 Cologne,

Germany

ARIBERT STILE (18), Institut far Biologie

III, Albert-Ludwigs-Universitiit Freiburg,

VAS1LY M STUDITSKY (19), Laboratory of

Molecular Biology, National Institute of Di-

abetes and Digestive and Kidney Diseases,

National Institutes of Health, Bethesda,

Maryland 20892

HENDRIK G STUNNENBERG (12), EMBL,

D-69117 Heidelberg, Germany

HONG TANG (37), Department of Chemistry

and Waksman Institute, Rutgers University,

New Brunswick, New Jersey 08855

DEAN TANTIN (11), Molecular Biology Insti-

tute, University of California, Los Angeles,

Los Angeles, California 90095

DIMITRIS THANOS (13), Department of Bio-

chemistry and Molecular Biophysics, Co-

lumbia University, New York, New York

10032

FRITZ THOMA (16), Institut far Zellbiologie,

EidgenOssische Technische Hochschule,

ETH-HOnggerberg, CH-8093 Zurich, Swit-

zerland

NANCY E THOMPSON (39), McArdle Labora-

tory for Cancer Research, University of

Wisconsin-Madison, Madison, Wisconsin

53706

TOSHIO TSUKIYAMA (23), Laboratory of Bio-

chemistry, National Cancer Institute, Na-

tional Institutes of Health, Bethesda, Mary-

land 20892

BRYAN M TURNER (15), Chromatin and Gene Expression Group, Anatomy Department, University of Birmingham Medical School, Edgbaston, Birmingham B15 2TT, United Kingdom

CHIHARU UEGUCHI (21), Laboratory of Mo- lecular Microbiology, School of Agricul- ture, Nagoya University, Nagoya 464, Japan

KIYOE URA (20), Laboratory of Molecular Embryology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland

20892

RHEA T UTLEY (22), Department of Bio- chemistry and Molecular Biology, Pennsyl- vania State University, University Park, Pennsylvania 16802

RAFAEL VALCARCEL (12), EMBL, D-69117 Heidelberg, Germany

PIETER VAN DE PUTI'E (4), Laboratory of Mo- lecular Genetics, Leiden Institute of Chem- istry, Leiden University, Gorlaeus Labora- tories, 2300 RA Leiden, The Netherlands

PETER VAN ULSEN (4), Laboratory of Molecu- lar Genetics, Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories,

2300 RA Leiden, The Netherlands

PETER H VON HIPPEL (30), Institute of Molec- Mar Biology, Department of Chemistry, University of Oregon, Eugene, Oregon 974O3

DAGUANG WANG (27), Department of Biol- ogy, Washington University, St Louis, Mis- souri 63130

WILLIAM WHALEN (29, 30), Laboratory of Molecular Virology, National Cancer Insti- tute, National Institutes of Health, Bethesda, Maryland 20892

JAN WISNIEWSKI (9), Laboratory of Biochem- istry, National Cancer Institute, National In- stitutes of Health, Bethesda, Maryland 2O892

ALAN P WOLFFE (20), Laboratory of Molecu- lar Embryology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland

20892

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x v i CONTRIBUTORS TO VOLUME 274

KRYSTYNA WOLSKA (30), Institute of Microbi-

ology, University of Warsaw, Warsaw 64,

Poland

JERRY L WORKMAN (22), Department of Bio-

chemistry and Molecular Biology and Cen-

ter for Gene Regulation, Pennsylvania State

University, University Park, Pennsylvania

16802

CARL WU (9, 23), Laboratory of Biochemis-

try, National Cancer Institute, National

20892

MOSHE YANIV (14), Unit~ des Virus Onco- gOnes, Ddpartement des Biotechnologies, Institut Pasteur, 75724 Paris, France

WEN-CHEN YEH (8), Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218

M1N ZHONG (9), Laboratory of Biochemistry, National Cancer Institute, National Insti- tutes of Health, Bethesda, Maryland 20892

LAURENCE ZULIANELLO (4), Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, 2300 RA Leiden, The Nether- lands

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M E T H O D S IN E N Z Y M O L O G Y

VOLUME I Preparation and Assay of Enzymes

Edited by SIDNEY P COLOWICK AND NATHAN 0 KAPLAN

VOLUME lI Preparation and Assay of Enzymes

VOLUME III Preparation and Assay of Substrates

Edited by SIDNEY P COLOWICK AND NATHAN O KAPLAN

VOLUME IV Special Techniques for the Enzymologist

VOLUME V Preparation and Assay of Enzymes

VOLUME VI Preparation and Assay of Enzymes (Continued)

Preparation and Assay of Substrates

Special Techniques

VOLUME VII Cumulative Subject Index

Edited by SIDNEY P COLOWlCK AND NATHAN O KAPLAN

VOLUME VIII Complex Carbohydrates

Edited by ELIZABETH F NEUFELD AND VICTOR GINSBURG

VOLUME IX Carbohydrate Metabolism

Edited by WILLIS A WOOD

VOLUME X Oxidation and Phosphorylation

Edited by RONALD W ESTABROOK AND MAYNARD E PULLMAN VOLUME XI Enzyme Structure

Edited by C H W HIRS

VOLUME XII Nucleic Acids (Parts A and B)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE

VOLUME XIII Citric Acid Cycle

Edited by J M LOWENSTEIN

VOLUME XIV Lipids

Edited by J M LOWENSTEIN

VOLUME XV Steroids and Terpenoids

Edited by RAYMOND B CLAYTON

VOLUME XVI Fast Reactions

Edited by KENNETH KUSTIN

xix

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VOLUME XVII Metabolism of Amino Acids and Amines (Parts A and B)

Edited by HERBERT TABOR AND CELIA WHITE TABOR

VOLUME XVIII Vitamins and Coenzymes (Parts A, B, and C)

Edited by DONALD B McCORMICK AND LEMUEL D WRIGHT

VOLUME XIX Proteolytic Enzymes

Edited by GERTRUDE E PERLMANN AND LASZLO LORAND

VOLUME XX Nucleic Acids and Protein Synthesis (Part C)

Edited by KIVtE MOLDAVE AND LAWRENCE GROSSMAN

VOLUME XXI Nucleic Acids (Part D)

VOLUME XXII Enzyme Purification and Related Techniques

Edited by WILLIAM B JAKOBY

VOLUME XXIII Photosynthesis (Part A )

Edited by ANTHONY SAN PIETRO

VOLUME XXIV Photosynthesis and Nitrogen Fixation (Part B)

VOLUME XXV Enzyme Structure (Part B)

Edited by C H W HIRS AND SERGE N TIMASHEEF

VOLUME XXVI Enzyme Structure (Part C)

Edited by C H W HIRS AND SERGE N TIMASHEFF

VOLUME XXVII Enzyme Structure (Part D)

Edited by C H W HIRS AND SERGE N TIMASHEFE

VOLUME XXVIII Complex Carbohydrates (Part B)

Edited by VICTOR GINSBURG

VOLUME XXIX Nucleic Acids and Protein Synthesis (Part E)

VOLUME XXX Nucleic Acids and Protein Synthesis (Part F)

Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN

VOLUME XXXI Biomembranes (Part A )

Edited by SIDNEY FLEISCHER AND LESTER PACKER

VOLUME XXXII Biomembranes (Part B)

VOLUME XXXIII Cumulative Subject Index Volumes I - X X X

Edited by MARTHA G DENNIS AND EDWARD A DENNIS

VOLUME XXXIV Affinity Techniques (Enzyme Purification: Part B)

Edited by WILLIAM B JAKOBY AND MEIR WILCHEK

VOLUME XXXV Lipids (Part B)

Edited by JOHN M LOWENSTEIN

VOLUME XXXVI Hormone Action (Part A: Steroid Hormones)

Edited by BERT W O'MALLEY AND JOEL G HARDMAN

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METHODS IN ENZYMOLOGY xxi

VOLUME XXXVII Hormone Action (Part B: Peptide Hormones)

VOLUME XXXVIII Hormone Action (Part C: Cyclic Nucleotides)

Edited by JOEL G HARDMAN AND BERT W O'MALLEY

VOLUME XXXIX Hormone Action (Part D: Isolated Cells, Tissues, and Organ Systems)

Edited by JOEL G HARDMAN AND BERT W O'MALLEY

VOLUME XL Hormone Action (Part E: Nuclear Structure and Function)

VOLUME XLI Carbohydrate Metabolism (Part B)

Edited by W A WOOD

VOLUME XLII Carbohydrate Metabolism (Part C)

Edited by W A Wood

VOLUME XLIII Antibiotics

Edited by JOHN H HASH

VOLUME XLIV Immobilized Enzymes

Edited by KLAUS MOSBACH

VOLUME XLV Proteolytic Enzymes (Part B)

Edited by LASZLO LORAND

VOLUME XLVI Affinity Labeling

Edited by WILLIAM B JAKOBY AND MEIR WILCHEK

VOLUME XLVII Enzyme Structure (Part E)

VOLUME XLVIII Enzyme Structure (Part F)

VOLUME XLIX Enzyme Structure (Part G)

VOLUME L Complex Carbohydrates (Part C)

VOLUME LI Purine and Pyrimidine Nucleotide Metabolism

Edited by PATRICIA A HOFFEE AND MARY ELLEN JONES

VOLUME LII Biomembranes (Part C: Biological Oxidations)

VOLUME LIII Biomembranes (Part D: Biological Oxidations)

VOLUME LIV Biomembranes (Part E: Biological Oxidations)

VOLUME KW Biomembranes (Part F: Bioenergetics)

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xxii METHODS IN ENZYMOLOGY

VOLUME LVI Biomembranes (Part G: Bioenergetics)

Edited by SIDNEY FLEISCHER AND LESTER PACKER

VOLUME LVII Bioluminescence and Chemiluminescence

Edited by MARLENE A DELUCA

VOLUME LVIII Cell Culture

Edited by WILLIAM B JAKOBY AND IRA PASTAN

VOLUME LIX Nucleic Acids and Protein Synthesis (Part G)

Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN

VOLUME LX Nucleic Acids and Protein Synthesis (Part H)

Edited by KIVlE MOLDAVE AND LAWRENCE GROSSMAN

VOLUME 61 Enzyme Structure (Part H)

Edited by C H W HIRS AND SERGE N TIMASHEFF

VOLUME 62 Vitamins and Coenzymes (Part D)

VOLUME 63 Enzyme Kinetics and Mechanism (Part A: Initial Rate and Inhibitor Methods)

Edited by DANIEL L PURICH

VOLUME 64 Enzyme Kinetics and Mechanism (Part B: Isotopic Probes and Com- plex Enzyme Systems)

VOLUME 65 Nucleic Acids (Part I)

VOLUME 66 Vitamins and Coenzymes (Part E)

VOLUME 67 Vitamins and Coenzymes (Part F)

VOLUME 68 Recombinant DNA

Edited by RAY Wu

VOLUME 69 Photosynthesis and Nitrogen Fixation (Part C)

VOLUME 70 Immunochemical Techniques (Part A)

Edited by HELEN VAN VUNAKIS AND JOHN J LANGONE

VOLUME 71 Lipids (Part C)

Edited by JOHN M LOWENSTEIN

VOLUME 72 Lipids (Part D)

Edited by JOHN M LOWENSTEIN

VOLUME 73 Immunochemical Techniques (Part B)

Edited by JOHN J LANGONE AND HELEN VAN VUNAKIS

VOLUME 74 lmmunochemical Techniques (Part C)

Edited by JOHN J LANGONE AND HELEN VAN VUNAKIS

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METHODS IN ENZYMOLOGY XXlll

VOLUME 75 Cumulative Subject Index Volumes XXXI, XXXII, XXXIV-LX

Edited by EDWARD A DENNIS AND MARTHA G DENNIS

VOLUME 76 Hemoglobins

Edited by ERALDO ANTONINI, LUIGI ROSsI-BERNARDI, AND EMILIA CHIANCONE VOLUME 77 Detoxication and Drug Metabolism

VOLUME 78 Interferons (Part A)

Edited by SIDNEY PESTKA

VOLUME 79 Interferons (Part B)

Edited by SIDNEY PESTKA

VOLUME 80 Proteolytic Enzymes (Part C)

Edited by LASZLO LORAND

VOLUME 81 Biomembranes (Part H: Visual Pigments and Purple Mem-

branes, I)

Edited by LESTER PACKER

VOLUME 82 Structural and Contractile Proteins (Part A: Extracellular Matrix)

Edited by LEON W CUNNINGHAM AND DIXIE W FREDERIKSEN

VOLUME 83 Complex Carbohydrates (Part D)

VOLUME 84 Immunochemical Techniques (Part D: Selected Immunoassays)

Edited by JOHN J LANGONE AND HELEN VAN VUNAKIS

VOLUME 85 Structural and Contractile Proteins (Part B: The Contractile Appara- tus and the Cytoskeleton)

Edited by DIXIE W FREDERIKSEN AND LEON W CUNNINGHAM

VOLUME 86 Prostaglandins and Arachidonate Metabolites

Edited by WILLIAM E M LANDS AND WILLIAM L SMITH

VOLUME 87 Enzyme Kinetics and Mechanism (Part C: Intermediates, Stereo- chemistry, and Rate Studies)

VOLUME 88 Biomembranes (Part I: Visual Pigments and Purple Mem-

branes, II)

VOLUME 89 Carbohydrate Metabolism (Part D)

VOLUMe 90 Carbohydrate Metabolism (Part E)

VOLUME 91 Enzyme Structure (Part I)

VOLUME 92 Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)

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xxiv METHODS IN ENZYMOLOGY

VOLUME 93 Immunochemical Techniques (Part F: Conventional Antibodies, Fc Receptors, and Cytotoxicity)

Edited by JOHN J LANGONE AND HELEN VAN VUNAK1S

VOLUME 94 Polyamines

Edited by HERBERT TABOR AND CELIA WHITE TABOR

VOLUME 95 Cumulative Subject Index Volumes 61-74, 76-80

Edited by EDWARD A DENNIS AND MARTHA G DENNIS

VOLUME 96 Biomembranes [Part J: Membrane Biogenesis: Assembly and Tar- geting (General Methods; Eukaryotes)]

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER

VOLUME 97 Biomembranes [Part K: Membrane Biogenesis: Assembly and Tar- geting (Prokaryotes, Mitochondria, and Chloroplasts)]

VOLUME 98 Biomembranes (Part L: Membrane Biogenesis: Processing and Re- cycling)

VOLUME 99 Hormone Action (Part F: Protein Kinases)

Edited by JACKIE D CORBIN AND JOEL G HARDMAN

VOLUME 100 Recombinant DNA (Part B)

Edited by RAY Wu, LAWRENCE GROSSMAN, AND KIVIE MOLDAVE

VOLUME 101 Recombinant DNA (Part C)

Edited by RAY W u , LAWRENCE GROSSMAN, AND KIVIE MOLDAVE

VOLUME 102 Hormone Action (Part G: Calmodulin and Calcium-Binding Pro- teins)

Edited by ANTHONY R MEANS AND BERT W O'MALLEY

VOLUME 103 Hormone Action (Part H: Neuroendocrine Peptides)

Edited by P MICHAEL CONN

VOLUME 104 Enzyme Purification and Related Techniques (Part C)

VOLUME 105 Oxygen Radicals in Biological Systems

Edited by LESTER PACKER

VOLUME 106 Posttranslational Modifications (Part A)

Edited by FINN WOLD AND KIVIE MOLDAVE

VOLUME 107 Posttranslational Modifications (Part B)

Edited by FINN WoLD AND KIVIE MOLDAVE

VOLUME 108 Immunochemical Techniques (Part G: Separation and Characteriza- tion of Lymphoid Cells)

Edited by GIOVANNI DI SABATO, JOHN J LANGONE, AND

HELEN VAN VUNAKIS

VOLUME 109 Hormone Action (Part I: Peptide Hormones)

Edited by LuTz BIRNBAUMER AND BERT W O'MALLEY

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METHODS IN ENZYMOLOGY XXV VOLUME 110 Steroids and Isoprenoids (Part A)

Edited by JOHN H LAW AND HANS C RILLING

VOLUME 111 Steroids and Isoprenoids (Part B)

Edited by JOHN H LAW AND HANS C R1LLING

VOLUME 112 Drug and Enzyme Targeting (Part A)

Edited by KENNETH J WIDDER AND RALPH GREEN

VOLUME 113 Glutamate, Glutamine, Glutathione, and Related Compounds

Edited by ALTON MEISTER

VOLUME 114 Diffraction Methods for Biological Macromolecules (Part A)

Edited by HAROLD W WYCKOFF, C H W HIRS, AND SERGE N TIMASHEFF VOLUME 115 Diffraction Methods for Biological Macromolecules (Part B)

Edited by HAROLD W WYCKOFF, C H W HIRS, AND SERGE N TIMASHEFF VOLUME 116 Immunochemical Techniques (Part H: Effectors and Mediators of Lymphoid Cell Functions)

Edited by GIOVANNI DI SABATO, JOHN J LANGONE, AND HELEN VAN

VUNAKIS

VOLUME 117 Enzyme Structure (Part J)

Edited by C H W HIRS AND SERGE N TIMASHEFF

VOLUME 118 Plant Molecular Biology

Edited by ARTHUR WEISSBACH AND HERBERT WEISSBACH

VOLUME 119 Interferons (Part C)

Edited by SIDNEY PESTKA

VOLUME 121 Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)

VOLUME 122 Vitamins and Coenzymes (Part G)

Edited by FRANK CHYTIL AND DONALD B McCoRMICK

VOLUME 123 Vitamins and Coenzymes (Part H)

Edited by FRANK CHYTIL AND DONALD B McCoRMICK

VOLUME 124 Hormone Action (Part J: Neuroendocrine Peptides)

VOLUME 125 Biomembranes (Part M: Transport in Bacteria, Mitochondria, and Chloroplasts: General Approaches and Transport Systems)

VOLUME 126 Biomembranes (Part N: Transport in Bacteria, Mitochondria, and Chloroplasts: Protonmotive Force)

VOLUME 127 Biomembranes (Part O: Protons and Water: Structure and Translo- cation)

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xxvi M E T H O D S I N E N Z Y M O L O G Y

VOLUME 128 Plasma Lipoproteins (Part A: Preparation, Structure, and Molecu- lar Biology)

Edited by JERE P SEGREST AND JOHN J ALBERS

VOLUME 129 Plasma Lipoproteins (Part B: Characterization, Cell Biology, and Metabolism)

Edited by JOHN J ALBERS AND JERE P SEGREST

VOLUME 130 Enzyme Structure (Part K)

Edited by C H W HIRS AND SERGE N TIMASHEFE

VOLUME 131 Enzyme Structure (Part L)

VOLUME 132 Immunochemical Techniques (Part J: Phagocytosis and Cell-Medi- ated Cytotoxicity)

Edited by GIOVANNI DI SABATO AND JOHANNES EVERSE

VOLUME 133 Bioluminescence and Chemiluminescence (Part B)

Edited by MARLENE DELuCA AND WILLIAM D MCELRoY

VOLUME 134 Structural and Contractile Proteins (Part C" The Contractile Appa- ratus and the Cytoskeleton)

Edited by RICHARD B VALLEE

VOLUME 135 Immobilized Enzymes and Cells (Part B)

VOLUME 136 Immobilized Enzymes and Cells (Part C)

VOLUME 137 Immobilized Enzymes and Cells (Part D)

VOLUME 138 Complex Carbohydrates (Part E)

VOLUME 139 Cellular Regulators (Part A: Calcium- and Calmodulin-Binding Proteins)

Edited by ANTHONY R MEANS AND P MICHAEL CONN

VOLUME 141 Cellular Regulators (Part B: Calcium and Lipids)

Edited by P MICHAEL CONN AND ANTHONY R MEANS

VOLUME 142 Metabolism of Aromatic Amino Acids and Amines

Edited by SEYMOUR KAUFMAN

VOLUME 143 Sulfur and Sulfur Amino Acids

Edited by WILLIAM B JAKOBY AND OWEN GRIFFITH

VOLUME 144 Structural and Contractile Proteins (Part D: Extracellular Matrix)

Edited by LEON W CUNNINGHAM

VOLUME 145 Structural and Contractile Proteins (Part E: Extracellular Matrix)

Edited by LEON W CUNNINGHAM

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METHODS IN ENZYMOLOGY xxvii VOLUME 146 Peptide Growth Factors (Part A)

Edited by DAVID BARNES AND DAVID A SIRBASKU

VOLUME 147 Peptide Growth Factors (Part B)

Edited by DAVID BARNES AND DAVID A S1RBASKU

VOLUME 148 Plant Cell Membranes

Edited by LESTER PACKER AND ROLAND DOUCE

VOLUME 149 Drug and Enzyme Targeting (Part B)

Edited by RALPH GREEN AND KENNETH J WIDDER

VOLUME 150 Immunochemical Techniques (Part K: In Vitro Models of B and T

Cell Functions and Lymphoid Cell Receptors)

Edited by GIOVANNI DI SABATO

VOLUME 151 Molecular Genetics of Mammalian Cells

Edited by MICHAEL M GOTTESMAN

VOLUME 152 Guide to Molecular Cloning Techniques

Edited by SHELBY L BERGER AND ALAN R KIMMEL

VOLUME 153 Recombinant D N A (Part D)

Edited by RAY Wu AND LAWRENCE GROSSMAN

VOLUME 154 Recombinant DNA (Part E)

Edited by RAY WU AND LAWRENCE GROSSMAN

VOLUME 155 Recombinant DNA (Part F)

Edited by RAY Wu

VOLUME 156 Biomembranes (Part P: ATP-Driven Pumps and Related Trans- port: The Na,K-Pump)

VOLUME 157 Biomembranes (Part Q: ATP-Driven Pumps and Related Trans- port: Calcium, Proton, and Potassium Pumps)

VOLUME 158 Metalloproteins (Part A)

Edited by JAMES F RIORDAN AND BERT L VALLEE

VOLUME 159 Initiation and Termination of Cyclic Nucleotide Action

Edited by JACKIE D CORBIN AND ROGER A JOHNSON

VOLUME 160 Biomass (Part A" Cellulose and Hemicellulose)

Edited by WILLIS A WOOD AND SCOTT T KELLOGG

VOLUME 161 Biomass (Part B: Lignin, Pectin, and Chitin)

Edited by WILLIS A WOOD AND SCOTT T KELLOGG

VOLUME 162 Immunochemical Techniques (Part L: Chemotaxis and Inflam- mation)

VOLUME 163 Immunochemical Techniques (Part M: Chemotaxis and Inflam- mation)

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XXVlll METHODSIN ENZYMOLOGY

VOLUME 164 Ribosomes

Edited by HARRY F NOLLER, JR., AND KIVIE MOLDAVE

VOLUME 165 Microbial Toxins: Tools for Enzymology

Edited by SIDNEY HARSHMAN

VOLUME 166 Branched-Chain Amino Acids

Edited by ROBERT HARRIS AND JOHN R SOKATCH

VOLUME 167 Cyanobacteria

Edited by LESTER PACKER AND ALEXANDER N GLAZER

VOLUME 168 Hormone Action (Part K: Neuroendocrine Peptides)

VOLUME 169 Platelets: Receptors, Adhesion, Secretion (Part A)

Edited by JACEK HAWlGER

VOLUME 170 N u c l e o s o m e s

Edited by PAUL M WASSARMAN AND ROGER D KORNBERG

VOLUME 171 Biomembranes (Part R: Transport Theory: Cells and Model Mem- branes)

VOLUME 172 Biomembranes (Part S: Transport: Membrane Isolation and Char- acterization)

Edited by SIDNEY FLE1SCHER AND BECCA FLEISCHER

VOLUME 173 Biomembranes [Part T: Cellular and Subcellular Transport: Eukary- otic (Nonepithelial) Cells]

VOLUME 174 Biomembranes [Part U: Cellular and Subcellular Transport: Eukar- yotic (Nonepithelial) Cells]

VOLUME 176 Nuclear Magnetic Resonance (Part A: Spectral Techniques and Dy- namics)

Edited by NORMAN J OPPENHEIMER AND THOMAS L JAMES

VOLUME 177 Nuclear Magnetic Resonance (Part B: Structure and Mechanism)

Edited by NORMAN J OPPENHEIMER AND THOMAS L JAMES

VOLUME 178 Antibodies, Antigens, and Molecular Mimicry

Edited by JOHN J LANGONE

VOLUME 179 Complex Carbohydrates (Part F)

Edited by VICTOR GINSBURG

VOLUME 180 RNA Processing (Part A: General Methods)

Edited by JAMES E DAHLBERG AND JOHN N ABELSON

VOLUME 181 RNA Processing (Part B: Specific Methods)

Edited by JAMES E DAHLBERG AND JOHN N ABELSON

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METHODS IN ENZYMOLOGY x x i x

VOLUME 182 Guide to Protein Purification

Edited by MURRAY P DEUTSCHER

VOLUME 183 Molecular Evolution: Computer Analysis of Protein and Nucleic Acid Sequences

Edited by RUSSELL F DOOLITTLE

VOLUME 184 Avidin-Biotin Technology

Edited by MEIR WILCHEK AND EDWARD A BAYER

VOLUME 185 Gene Expression Technology

Edited by DAVID V GOEDDEL

VOLUME 186 Oxygen Radicals in Biological Systems (Part B: Oxygen Radicals and Antioxidants)

Edited by LESTER PACKER AND ALEXANDER N GLAZER

VOLUME 187 Arachidonate Related Lipid Mediators

Edited by ROBERT C MURPHY AND FRANK A FITZPATRICK

VOLUME 188 Hydrocarbons and Methylotrophy

Edited by MARY E LIDSTROM

VOLUME 189 Retinoids (Part A: Molecular and Metabolic Aspects)

VOLUME 190 Retinoids (Part B: Cell Differentiation and Clinical Applications)

VOLUME 191 Biomembranes (Part V: Cellular and Subcellular Transport: Epithe- lial Cells)

VOLUME 192 Biomembranes (Part W: Cellular and Subcellular Transport: Epi- thelial Cells)

VOLUME 193 Mass Spectrometry

Edited by JAMES A McCLOSKEY

VOLUME 194 Guide to Yeast Genetics and Molecular Biology

Edited by CHmSTINE GUTHRIE AND GERALD R FINK

VOLUME 195 Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase

Edited by ROGER A JOHNSON AND JACKIE D CORBIN

VOLUME 196 Molecular Motors and the Cytoskeleton

Edited by RICHARD B VALLEE

VOLUME 197 Phospholipases

Edited by EDWARD A DENNIS

VOLUME 198 Peptide Growth Factors (Part C)

Edited by DAVID BARNES, J P MATHER, AND GORDON H SATO

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XXX METHODS IN ENZYMOLOGY

VOLUME 200 Protein Phosphorylation (Part A: Protein Kinases: Assays, Purifica- tion, Antibodies, Functional Analysis, Cloning, and Expression)

Edited by TONY HUNTER AND BARTHOLOMEW M SEFrON

VOLUME 201 Protein Phosphorylation (Part B: Analysis of Protein Phosphoryla- tion, Protein Kinase Inhibitors, and Protein Phosphatases)

Edited by TONY HUNTER AND BARTHOLOMEW M SEFTON

VOLUME 202 Molecular Design and Modeling: Concepts and Applications (Part A: Proteins, Peptides, and Enzymes)

VOLUME 203 Molecular Design and Modeling: Concepts and Applications (Part B: Antibodies and Antigens, Nucleic Acids, Polysaccharides, and Drugs)

VOLUME 204 Bacterial Genetic Systems

Edited by JEFFREY H MILLER

VOLUME 205 Metallobiochemistry (Part B: Metallothionein and Related Mole- cules)

Edited by JAMES F RIORDAN AND BERT L VALLEE

VOLUME 206 Cytochrome P450

Edited by MICHAEL R WATERMAN AND ERIC F JOHNSON

VOLUME 207 Ion Channels

Edited by BERNARDO RUDY AND LINDA E IVERSON

VOLUME 208 P r o t e i n - D N A I n t e r a c t i o n s

Edited by ROBERT T SAUER

VOLUME 209 Phospholipid Biosynthesis

Edited by EDWARD A DENNIS AND DENNIS E VANCE

VOLUME 210 Numerical Computer Methods

Edited by LUDWIG BRAND AND MICHAEL L JOHNSON

VOLUME 211 D N A Structures (Part A: Synthesis and Physical Analysis of DNA)

Edited by DAVID M J LILLEY AND JAMES E DAHLBERG

VOLUME 212 DNA Structures (Part B: Chemical and Electrophoretic Analysis

of DNA)

Edited by DAVID M J LILLEY AND JAMES E DAHLBERG

VOLUME 213 Carotenoids (Part A: Chemistry, Separation, Quantitation, and Antioxidation)

VOLUME 214 Carotenoids (Part B: Metabolism, Genetics, and Biosynthesis)

VOLUME 215 Platelets: Receptors, Adhesion, Secretion (Part B)

Edited by JACEK J HAWIGER

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METHODS IN ENZYMOLOGY xxxi VOLUME 216 Recombinant DNA (Part G)

VOLUME 219 Reconstitution of Intracellular Transport

Edited by JAMES E ROTHMAN

VOLUME 220 Membrane Fusion Techniques (Part A)

Edited by NEJAT DCrZGONE~

VOLUME 221 Membrane Fusion Techniques (Part B)

Edited by NEJAT DC3ZOONE~

VOLUME 222 Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part A: Mammalian Blood Coagulation Factors and Inhibitors)

Edited by LASZLO LORAND AND KENNETH G MANN

VOLUME 223 Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part B: Complement Activation, Fibrinolysis, and Nonmammalian Blood Coagulation Factors)

Edited by LASZLO LORAND AND KENNETH G MANN

VOLUME 224 Molecular Evolution: Producing the Biochemical Data

Edited by ELIZABETH ANNE ZIMMER, THOMAS J WHITE, REBECCA L CANN, AND ALLAN C WILSON

VOLUME 225 Guide to Techniques in Mouse Development

Edited by PAUL M WASSARMAN AND MELVIN L DEPAMPHILIS

VOLUME 226 Metallobiochemistry (Part C: Spectroscopic and Physical Methods for Probing Metal Ion Environments in Metalloenzymes and Metalloproteins)

Edited by JAMES F RIORDAN AND BERT L VALLEE

VOLUME 227 Metallobiochemistry (Part D: Physical and Spectroscopic Methods for Probing Metal Ion Environments in Metalloproteins)

Edited by JAMES F RIORDAN AND BERT L VALLEE

VOLUME 228 Aqueous Two-Phase Systems

Edited by HARRY WALTER AND GOTE JOHANSSON

VOLUME 230 Guide to Techniques in Glycobiology

Edited by WILLIAM J LENNARZ AND GERALD W HART

VOLUME 231 Hemoglobins (Part B: Biochemical and Analytical Methods)

Edited by JOHANNES EVERSE, KIM D VANDEGRIFF, AND ROBERT M WlNSLOW

VOLUME 232 Hemoglobins (Part C: Biophysical Methods)

Edited by JOHANNES EVERSE, KIM D VANDEGRIFF, AND ROBERT M WINSLOW

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xxxii METHODS IN ENZYMOLOGY

VOLUME 233 Oxygen Radicals in Biological Systems (Part C)

VOLUME 234 Oxygen Radicals in Biological Systems (Part D)

VOLUME 235 Bacterial Pathogenesis (Part A: Identification and Regulation of Virulence Factors)

Edited by VIRGINIA L CLARK AND PATRIK M BAVO1L

VOLUME 236 Bacterial Pathogenesis (Part B: Integration of Pathogenic Bacteria with Host Cells)

Edited by VIRGINIA L CLARK AND PATRIK M BAVOIL

VOLUME 237 Heterotrimeric G Proteins

Edited by RAVI IYENGAR

VOLUME 238 Heterotrimeric G-Protein Effectors

Edited by RAVI IYENGAR

VOLUME 239 Nuclear Magnetic Resonance (Part C)

Edited by THOMAS L JAMES AND NORMAN J OPPENHEIMER

VOLUME 240 Numerical Computer Methods (Part B)

Edited by MICHAEL L JOHNSON AND LUDWIG BRAND

VOLUME 241 Retroviral Proteases

Edited by LAWRENCE C KUO AND JULES A SHAEER

VOLUME 242 Neoglycoconjugates (Part A)

Edited by Y C LEE AND REIKO T LEE

VOLUME 243 Inorganic Microbial Sulfur Metabolism

Edited by HARRY D PECK, JR., AND JEAN LEGALL

VOLUME 244 Proteolytic Enzymes: Serine and Cysteine Peptidases

Edited by ALAN J BARRETT

VOLUME 245 Extracellular Matrix Components

Edited by E RUOSLAHTI AND E ENGVALL

VOLUME 246 Biochemical Spectroscopy

Edited by KENNETH SAUER

VOLUME 247 Neoglycoconjugates (Part B: Biomedical Applications)

Edited by Y C LEE AND REIKO T LEE

VOLUME 248 Proteolytic Enzymes: Aspartic and Metallo Peptidases

Edited by ALAN J BARREq"F

VOLUME 249 Enzyme Kinetics and Mechanism (Part D: Developments in En- zyme Dynamics)

VOLUME 250 Lipid Modifications of Proteins

Edited by PATRICK J CASEY AND JANICE E B u s s

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VOLUME 251 Biothiols (Part A: Monothiols and Dithiols, Protein Thiols, and Thiyl Radicals)

VOLUME 252 Biothiols (Part B: Glutathione and Thioredoxin; Thiols in Signal Transduction and Gene Regulation)

VOLUME 253 Adhesion of Microbial Pathogens

Edited by RON J DOYLE AND ITZHAK OFEK

VOLUME 254 Oncogene Techniques

Edited by PETER K VOGT AND INDER M VERMA

VOLUME 255 Small GTPases and Their Regulators (Part A" Ras Family)

Edited by W E BALCH, CHANNING J DER, AND ALAN HALL

VOLUME 256 Small GTPases and Their Regulators (Part B: Rho Family)

VOLUME 257 Small GTPases and Their Regulators (Part C: Proteins Involved in Transport)

VOLUME 258 Redox-Active Amino Acids in Biology

Edited by JUDITH P KLINMAN

VOLUME 259 Energetics of Biological Macromolecules

Edited by MICHAEL L JOHNSON AND GARY K ACKERS

VOLUME 260 Mitochondrial Biogenesis and Genetics (Part A)

Edited by GIUSEPPE M ATTARDI AND ANNE CHOMYN

VOLUME 261 Nuclear Magnetic Resonance and Nucleic Acids

Edited by THOMAS L JAMES

VOLUME 262 D N A Replication

Edited by JUDITH L CAMPBELL

VOLUME 263 Plasma Lipoproteins (Part C: Quantitation)

Edited by WILLIAM A BRADLEY, SANDRA H GIANTURCO, AND JERE P SEGREST VOLUME 264 Mitochondrial Biogenesis and Genetics (Part B)

Edited by GIUSEPPE M ATTARDI AND ANNE CHOMYN

VOLUME 265 Cumulative Subject Index Volumes 228, 230-262

VOLUME 266 Computer Methods for Macromolecular Sequence Analysis

Edited by RUSSELL F DOOLITTLE

VOLUME 267 Combinatorial Chemistry

Edited by JOHN N ABELSON

VOLUME 268 Nitric Oxide (Part A: Sources and Detection of NO; NO Synthase)

VOLUME 269 Nitric Oxide (Part B: Physiological and Pathological Processes)

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xxxiv M E T H O D S IN E N Z Y M O L O G Y

VOLUME 270 High Resolution Separation and Analysis of Biological Macromole- cules (Part A: Fundamentals)

Edited by BARRY L KARGER AND WILLIAM S HANCOCK

VOLUME 271 High Resolution Separation and Analysis Of Biological Macromole- cules (Part B: Applications)

Edited by BARRY L KARGER AND WILLIAM S HANCOCK

VOLUME 272 Cytochrome P450 (Part B)

Edited by ERIC F JOHNSON AND MICHAEL R WATERMAN

VOLUME 273 RNA Polymerase and Associated Factors (Part A)

Edited by SANKAR ADHYA

VOLUME 274 RNA Polymerase and Associated Factors (Part B)

Edited by SANKAR ADHYA

VOLUME 275 Viral Polymerases and Related Proteins

Edited by LAWRENCE C KUO, DAVID B OLSEN, AND STEVENS CARROLL VOLUME 276 Macromolecular Crystallography (Part A) (in preparation)

Edited by CHARLES W CARTER, JR., AND ROBERT M SWEET

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[1] S-150-DEPENDENT in Vitro TRANSCRIPTION ASSAY 3

[1] Regulated Transcription in a Complete Ribosome-Free

in Vitro System of E s c h e r i c h i a coli

By HYON E CHOY

The prokaryotic cell-free protein synthesis system that was developed

by Matthaei and Nirenberg I and perfected by Zubay (reviewed in Zubay 2) has greatly contributed to the elucidation of the mechanism of gene expression and its regulation Basically, it is the supernatant fraction (S-30) of 30,000 g centrifugation of lysed cells that contains most of the components for transcription and translation Thus, in this coupled transcription- translation system, protein products of an exogenously added D N A template are analyzed In studying transcription regulation, however, it is sim- pler to analyze nascent R N A rather than protein products A new method has been developed in which cell extract (S-150)-driven DNA-dependent

R N A synthesis can be directly monitored The S-150 is the supernatant of 150,000 g centrifugation of the previously mentioned S-30, and it is devoid

of ribosomes and the membrane fraction The S-150 of Escherichia coli has

been proven to contain various cytosolic proteins as well as R N A polymerase (see Results) The S-150-dependent transcription system is easy to prepare and yet it is effective in studying transcription regulation, especially when unidentified cytosolic components are involved

The cells are washed twice in a buffer containing 10 mM Tris-acetate,

pH 8.0, 15 m M magnesium acetate, 60 mM potassium acetate, 1 m M dithi-

1 j H M a t t h a e i a n d M N i r e n b e r g , Proc Natl Acad Sci U.S.A 47, 1 5 8 0 (1961)

2 G Z u b a y , A n n u Rev Genet 7, 2 6 7 ( 1 9 7 3 )

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4 BASAL TRANSCRIPTION AND REGULATION OF TRANSCRIPTION [ 1]

othreitol (DTI'), and 75/zg/ml pTSF The cell suspension (4 mg of cell/ml

of buffer) is lysed by a single pass through a French pressure cell at 6000 psi Immediately after lysis, DTT is again added to a final concentration of

1 mM The lysate is centrifuged at 30,000 g at 4 ° for 30 min The supernatant fraction is taken quickly and the 30,000 g centrifugation step is repeated once more The protein concentration of the lysate (S-30) is adjusted to

- 1 5 mg/ml and the lysate is dialyzed against the same buffer for 3 hr at

4 ° with two changes The S-30 lysate is then centrifuged at 150,000 g (65,000 rpm for 24 min with a table-top Optima TLX Ultracentrifuge, Beckman) The supernatant fraction (S-150) is collected and rapidly frozen in acetone- dry ice and stored at - 8 0 °

In Vitro Reactions

A typical reaction mixture contains transcription buffer (20 m M Tris- acetate, pH 8.0; 10 m M magnesium acetate; 100 mM potassium glutamate),

2 nM D N A template, 1 m M ATP, 0.1 m M GTP, 0.1 m M CTP, 0.01 m M UTP, 10-20 tzCi of [o~-32p]UTP (1 Ci = 37 GBq), and 1 unit RNasin in a total volume of 50/xl Note that for every promoter to be studied in vitro

using S-150, it is essential to optimize the salt condition, especially for Mg 2+ and K + (see Results) The reaction mixture is preincubated at 37 ° for - 5 min and the transcription reaction is initiated by the addition of S-150 to about 2 mg protein/ml The transcription reaction is terminated typically after a 6-min incubation at 37 ° by the addition of an equal volume of phenol : chloroform : isoamyl alcohol mixture (25 : 24 : 1) The mixture is vor- texed and centrifuged The aqueous phase is taken and treated again with phenol :chloroform:isoamyl alcohol The aqueous phase is taken and passed through a gel-filtration column (TE Micro Select-D, G-25, 5 Prime

3 Prime, Inc.) The gel-filtration step is included to remove small molecules bound to radioactive nucleotide, and thus cleans up the background on the gel The eluate is mixed with an equal volume of R N A loading buffer [80% (v/v) deionized formamide/1 x TBE (89 m M Tris-borate/2 m M EDTA); 0.025% (w/v) bromphenol blue; 0.025% (w/v) xylene cyanol] The mixture

is heated at 90 ° for 2 min and electrophoresed on a 8% polyacrylamide

D N A sequencing gel containing 8 M urea (40 cm long x 0.4 mm thick)

Results

S-150 is a cell extract generally devoid of membrane and ribosome material As shown earlier, the preparation of S-150 is relatively simple, unlike that of S-30 for the coupled transcription-translation reaction This

is probably because the translational apparatus consisting of multiple com-

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[1] S-150-DEPENDENT in Vitro TRANSCRIPTION ASSAY 5

v -5 SCHEME I

ponents is unstable In contrast, basic transcription activity requires only

R N A polymerase, a stable oligomeric protein It should be noted that in the process for S-150 preparation there is no particular step to remove endogenous DNA The bulk of endogenous DNA, however, is removed along with the membrane fraction by the 30,000-g centrifugation Most important, we think that the S-150 prepared by this procedure retains most

of the DNA-binding proteins (transcription factors), as far as we have tested (see below)

For an assay of any promoter it is necessary to optimize the salt condition, particularly for K + and Mg2+ 3 The type and the concentration of counter anion may also need to be varied, although 200 m M glutamate is optimal for most of the promoters 4,5 The promoter (DNA) must also be titrated in the presence of a fixed amount of S-150 (routinely about 2 mg/ml)

to carry out the reaction at a saturating concentration of the promoter Most

of the promoters tested saturate at about 2 nM in the presence of 2 mg/ml

of S-150 A concentration of S-150 higher than 2 mg/ml is not advised because it results in degradation of R N A (see below)

We examined the regulation of E coli gal operon expression using the S-150-dependent transcription assay (see Scheme I) The E coli gal operon

is driven by two partially overlapping promoters, gaIP1 and galP2 6-8 Tran- scription from P2 initiates 5 bp upstream of P1 c A M P - C R P complex binding at -41.5 from the transcription start site of P1 (+1) differentially modulates these gal promoters: P1 is activated while P2 is repressed by

c A M P - C R P 9'1° In studying the regulation of these gal promoters in vitro,

3 K A Jacobs and D Schlessinger, Biochemistry 16, 914 (1977)

4 S Leirmo, S C Harrison, D S Cayley, R R Burgess, and M T Record, Jr., Biochemistry

26, 2101 (1987)

5 H Choy, PhD thesis, University of California, Davis, 1989

6 R E Musso, R DiLauro, S Adhya, and B de Crombrugghe, Cell 12, 847 (1977)

7 H Aiba, S Adhya, and B de Crombrugghe, J Biol Chem 265, 11905 (1981)

8 S Adhya and W Miller, Nature (London) 279, 492 (1979)

9 M Irani, L Orosz, and S Adhya, Cell 32, 783 (1983)

10 H Choy and S Adhya, Proc Natl Acad Sci U.S.A 90, 472 (1993)

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6 BASAL TRANSCRIPTION AND REGULATION OF TRANSCRIPTION [ l ]

(pSA508) The pSA508 was derived from pIBI24 (International Biotechnologies, New Haven,

CT) The gal fragment in the plasmid was followed by a 54-bp Rho-independent transcription terminator (Ter) of the rpoC gene of E coli, the last stem and loop sequence Amp, bla gene;

Ori, origin of replication; R N A I, antisense RNA from Ori (see text)

we used a pBR322 type plasmid, pSA5091°, containing the gal promoter segment as a D N A template (Fig 1) In this plasmid a transcription terminator was placed after the gal promoters to detect gal transcripts of distinct small sizes

Figure 2 shows the results of various in vitro transcription reactions on

a denaturing polyacrylamide gel (8%) The first lane (left, Fig 2) shows the result of an in vitro transcription reaction using a purified R N A polymerase P1 and P2 labels with arrows indicate 120- and 125-nucleotide-long transcripts originated from galP1 and galP2, respectively Note that an equal intensity of transcripts from galP1 and galP2 was obtained A 108- nucleotide-long R N A I transcript was also detected The R N A I is an antisense repressor of replication of pBR322 and other ColE1 type plasmids laa2 A set of seven lanes in Fig 2 (middle) shows the result of a time course experiment with S-150 (6 mg/ml) The first lane (Fig 2) (after 2.5 min) shows the transcripts originating from galP1 and galP2 as well as the

R N A I However, a degradation of the transcripts was already noticed at 2.5 min R N A I transcripts of a multiplicity of lengths were detected: 103- nucleotide-long degradation product (the major product) as well as 108- nucleotide-long unprocessed R N A I (indicated by arrow in Fig 2) and visible intermediates Ribonuclease E, an E coli endoribonuclease, has been implicated in the removal of 5 nucleotides from the 5' end of R N A

11 S Lin-Chao and S N Cohen, Cell 65, 1233 (1991)

12 T Tomcsanyi and D Apirion, J Mol Biol 185, 713 (1985)

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[1] S - 1 5 0 - D E P E N D E N T in Vitro TRANSCRIPTION ASSAY 7

1.12 A s t h e i n c u b a t i o n t i m e i n c r e a s e d , we d e t e c t e d a f u r t h e r d e c r e a s e in

t h e f u l l - l e n g t h R N A f r o m b o t h gal p r o m o t e r s a n d R N A I, w h i c h was

a c c o m p a n i e d b y a n i n c r e a s e i n t h e d e g r a d a t e d p r o d u c t s T h e galP1 a n d

f o r m o s t of t h e assays t h e i n c u b a t i o n t i m e did n o t e x c e e d 6 min

T h e last t w o l a n e s i n Fig 2 show t r a n s c r i p t i o n i n t h e a b s e n c e o r p r e s e n c e

of 0.2 m M c A M P T h e i n c u b a t i o n t i m e was k e p t to 6 m i n a n d t h e a m o u n t

Trang 29

8 BASAL TRANSCRIPTION AND REGULATION OF TRANSCRIPTION [ 1]

of S-150 was reduced to 2 mg/ml to minimize the R N A degradation In the absence of cAMP about equal amounts of P1 and P2 R N A were detected However, on addition of cAMP, the galP1 R N A disappeared

while P2 R N A increased about threefold Interestingly, this result is identi- cal to that obtained with a purified in vitro system: P1 and P2 R N A were

produced equally in the absence of cAMP (first lane, Fig 2) but P1 R N A was elevated about threefold while P2 was undetectable in the presence of cAMP 12 Thus, we concluded that the S-150 contains (1) no endogenous cAMP and (2) sufficient CRP (cAMP receptor protein) molecules The dialysis of S-30 before the 150,000 g centrifugation must be effective in removing small molecules such as cAMP Most important, a transcription regulatory protein such as CRP is retained in the S-150, suggesting that transcription regulation may be studied using the S-150 In fact, we have identified a soluble factor required for the repression of gal operon expres-

sion in addition to the operon-specific repressor, GalR (observed by T Aki, H Choy, and S Adhya) Purified GalR represses the transcription from both gal promoters in vitro only in the presence of S-150.13 Starting from the S-150, we have successfully isolated a soluble factor by fraction- ation of the S-150.15

Although further improvement can be made by modifying E coli strains,

such as inactivating genes encoding nucleases or proteases, or by adding biologically relevant salts, the procedure described in this chapter serves

as a basis for cell extract-dependent in vitro transcription system This

system is also useful for studying ribonucleases in vitro, especially examining

their effect on nascent RNA Whereas the current system of wild-type E

coli requires a closed circular D N A template, a linear D N A fragment

could be used if the S-150 is prepared from a strain carrying mutations in exonuclease genes such as recD, xthA This system has been developed for

E coli but should also be applicable to other bacterial systems In the case

of Salmonella typhimurium, the only variation from the standard procedure

may be growing bacteria at 28° s

13 H Choy and S Adhya, Proc Natl Acad Sci U.S.A 89, 11264 (1992)

14 H Choy and S Adhya, Proc Natl Acad Sci U.S.A 90, 472 (1993)

is T Aki, H E Choy, and S Adhya, L 179 (1996)

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[2] SINGLE-STRANDED DNA-BINDING PROTEINS 9

[21 S i n g l e - S t r a n d e d D N A - B i n d i n g P r o t e i n s a s

T r a n s c r i p t i o n a l A c t i v a t o r s

By A L I T A MILLER, XING D A I , MIEYOUNG CHOI,

M A L E X A N D R A G L U C K S M A N N - K u I s , and LUCIA B ROTHMAN-DENES

Coliphage N4 uses the sequential activity of three different DNA-dependent RNA polymerases to transcribe its 72-kb linear, double-stranded DNA genome I A 320-kDa virion RNA polymerase, which is injected into the host cell along with the phage genome, is responsible for transcription of the early genes 2 Three early gene products are responsible for N4 middle transcription 3'4 Two of these proteins (30 and 40 kDa) constitute the hetero- dimeric N4 RNA polymerase 115; the third (17 kDa) polypeptide is required for promoter recognition by N4 RNA polymerase 11 6 Late transcription

is carried out by the host RNA polymerase 7 Our studies of the mechanism

of bacteriophage N4 early and late transcription led us to the finding that single-stranded DNA-binding proteins are required for transcriptional acti-

vation Escherichia coli single-stranded DNA-binding protein (EcoSSB)

activates N4 early promoters for recognition by N4 virion RNA polymerase 8 The phage-encoded, single-stranded DNA-binding protein (N4SSB)

is the transcriptional activator of E coli RNA polymerase at N4 late promoters 9 EcoSSB and N4SSB activate transcription by drastically different

mechanisms involving features specific to each protein, rather than those properties common to all single-stranded DNA-binding proteins, t° This chapter describes the procedures for purification of N4 virion RNA poly-

merase and N4 single-stranded DNA-binding protein, in vitro transcription

assays, characterization of the DNA structure at N4 early promoters, and

investigation of the interaction between EcoSSB and promoter-containing,

single-stranded templates

t D R Kiino and L B Rothman-Denes, in "The Bacteriophages" (R Calendar, ed.),

p 457 Plenum Press, New York, 1988

2 S C Falco, K VanderLaan, and L B Rothman-Denes, Proc Natl Acad Sci U.S.A 74,

520 (1977)

3 S C Falco and L B Rothman-Denes, Virology 95, 454 (1979)

4 W A Zehring, S C Falco, C Malone, and L B Rothman-Denes, Virology 126, 678 (1983)

5 W A Zehring and L B Rothman-Denes, J Biol Chem 258, 8074 (1983)

6 K Abravaya and L B Rothman-Denes, J Biol Chem 264, 12695 (1989)

7 R Zivin, W A Zehring, and L B Rothman-Denes, J Mol Biol 152, 335 (1981)

8 p Markiewicz, C Malone, J W Chase, and L B Rothman-Denes, Genes Dev 6, 2010 (1992)

9 N.-Y Cho, M Choi, and L B Rothman-Denes, J Mol Biol 246, 461 (1995)

10 j W Chase and K R Williams, Annu Rev Biochem 55, 130 (1986)

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10 BASAL TRANSCRIPTION AND REGULATION OF TRANSCRIPTION [9.] Role of EcoSSB in N4 E a r l y T r a n s c r i p t i o n

N4 early transcription requires the activity of two host factors, E c o S S B

utilize its promoters on either linear or supercoiled, double-stranded templates, 8,n but can transcribe denatured or single-stranded promoter-containing templates accurately and efficiently, le I n vitro, the addition of

transcription 8 Activation is specific to E c o S S B ; other single-stranded DNA- binding proteins (T4 gp32, T7 gp2.5, N4SSB, fd gpV) cannot substitute 8'13 Therefore, E c o S S B does not activate N4 early transcription merely through its ability to bind single-stranded DNA

Mutational analysis of two early promoters resident on single-stranded

D N A indicated that all determinants of N4 virion R N A polymerase-promoter recognition exist in the template strand and include specific sequences and a set of inverted repeats (Fig 1B) 14 These results suggested that N4 virion R N A polymerase recognizes a hairpin structure consisting of a 5-

to 7-bp stem and 3 base loop at the promoter Using single-stranded DNA- specific chemical and enzymatic probes, as well as T7 endonuclease I, which cleaves at D N A four-way junctions, 15 we have shown that, in the presence

of Mg e÷, the small D N A hairpins at N4 early promoters extrude at physiological superhelical densities (Fig 1 A ) ) 6 The hairpin on the template strand

is unusually stable and its loop is not reactive to single-stranded probes (Fig 1A), due to the base composition of the hairpin loop and the closing base pair 16 DNase I, neocarzinostatin, and N e u r o s p o r a crassa nuclease footprinting of promoter-containing, single-stranded DNAs indicate that

hairpin Instead, it stabilizes the template hairpin by binding to the adjacent single-stranded D N A (Fig 1B) In contrast, the complementary strand hairpin is disrupted 13 Therefore, we propose that E c o S S B activates N4 early promoters by providing an active promoter conformation, i.e., a stable

D N A hairpin required for N4 virion R N A polymerase promoter recogni-

t i o n ) 3 Other single-stranded DNA-binding proteins disrupt the template strand hairpin, indicating that the specificity of E c o S S B activation arises from its unique mode of interaction with the template strand hairpin DNA

n S C Falco, R Zivin, and L B Rothmann-Denes, Proc Natl Acad Sct U.S.A 75, 3220 (1978)

12 L L Haynes and L B Rothman-Denes, Cell 41, 597 (1985)

a3 M A Glucksmann-Kuis, X Dai, P Markiewicz, and L B Rothman-Denes, Cell 84, 147 (1996)

14 M m Glucksmann, P Markiewicz, C Malone, and L B Rothman-Denes, Cell 70, 491 (1992) x5 B de Massy, R A Weisberg, and F W Studier, J Mol Biol 193, 359 (1987)

16 X Dai, M Greizerstein, and L B Rothman-Denes, submitted (1996)

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[ Q , ] SINGLE-STRANDED DNA-BINDING PROTEINS 11

Trang 33

12 BASAL TRANSCRIPTION AND REGULATION OF TRANSCRIPTION [2] places the heparin-Sepharose 4B column and a Pharmacia (Piscataway,

N J) fast protein liquid chromatography (FPLC) system is used Purified N4 virion RNA polymerase is stored at - 8 0 °

Virion RNA Polymerase Transcription Assays in Vitro

Transcription reaction mixtures (100/xl) contain 10 m M Tris-HC1, pH 8.0, 10 mM MgC12,50 m M NaC1, 1 m M EDTA, 1 mM dithiothreitol (DTI'),

1 unit//xl RNasin (Promega, Madison, WI), 1 m M ATP, 1 m M GTP, 1 m M UTP (or CTP), 0.1 m M CTP (or UTP), 2-10 /zCi of [oz-32p]CTP (or [oe-32p]UTP)12 and DNA template Single-stranded templates are generated

by cloning promoter-containing fragments into the HinclI site of the sym-

metrical, multiple cloning site of M13mp7 Reannealing of the symmetrical cloning sites allows restriction of the single-stranded viral DNA, containing

the template strand of the promoter, by BamHI o r E c o R I 14 BamHI-re-

stricted single-stranded M13mp7 DNA (136 nucleotides in length) containing N4 early promoters (5 txg) is used in runoff transcription reactions When double-stranded, supercoiled templates (1/.~g) are used, these contain the N4 early promoters followed downstream by transcription termina-

tors 8,a6 EcoSSB (US Biochemicals, Cleveland, OH) is added to the desired EcoSSB/DNA (w/w) ratios Reactions are incubated for 5 min at 37 ° and

are terminated by the addition of E D T A and tRNA to final concentrations

of 5 m M and 100/xg/ml, respectively, followed by phenol extraction The samples are ethanol-precipitated, resuspended in loading buffer (80% formamide, 50 m M Tris-HC1, pH 8.0, 20 mM EDTA, and 0.5% each of bromophenol blue and xylene cyanol), and run on 8% (w/v) polyacrylamide/

7 M urea gels

Construction of Minicircle-Producing Plasmids and Isolation

of Minicircles

In order to study the effect of superhelical density on hairpin extrusion

at the N4 early promoters, we use a system that generates minicircles containing primarily N4 DNA sequences This approach isolates N4 DNA sequences from plasmid sequences that contain inverted repeats that might extrude in preference to the promoter hairpins and change the superhelical

density of the remaining sequences A 2.1-kb SpeI-PstI fragment of the

N4 genome, containing the N4 early promoters P1 and P2, followed by their natural terminators tl and t2,18 w a s cloned into plasmid pKB652I at

18 S Hattingh Willis and L B Rothman-Denes, unpublished (1996)

Trang 34

[2] SINGLE-STRANDED DNA-BINDING PROTEINS 13 the compatible XbaI and PstI sites to yield pXD102.16 pKB652I is a derivative of pKB652,19 which contains the polylinker region of M13mpl9 cloned

at the KpnI and HindlII sites The multiple cloning site is flanked by the

A attL and attR sites

In E coli KB204,19 which contains a defective A prophage with clts857 controlling the expression of the int and xis genes, site-specific recombination between A attL and attR sites of pXD102 yields two types of D N A circles: 2.1-kb circles, containing the inserted N4 sequences, and 3-kb vector circles To isolate the 2.1-kb circles, a 50-ml overnight culture of KB204 harboring pXD102 is diluted into 3 liters of LB containing 50/xg/

ml ampicillin and is incubated at 33 ° in a Lab-line/S.M.S hi-density fermentor (Lab-line Instruments, Melrose Park, IL) to an OD600 of 0.8-1.0 The growth temperature is increased to 42 ° within 10 min and the culture

is shaken at 42 ° for 3 hr Under these conditions, 70% of the pXD102 molecules in the culture undergo recombination Plasmid DNAs are isolated using Qiagen columns (Qiagen, Inc., Chatsworth, CA), digested

at 37 ° for 4 hr with EcoRI (5000 units/mg of total DNA), which linearizes only parent and vector circles, and analyzed on an agarose gel to ensure complete digestion The digested mixture is then banded on a CsC1 gradient (p = 1 g/ml) and the lower band, containing the supercoiled minicircles, is recovered

To prepare topoisomers of defined superhelical densities, D N A (6/zg)

is incubated with calf thymus topoisomerase I in the presence of varying concentrations of ethidium bromide (0-60 m M ) and 50 m M Tris-HC1,

pH 7.5, 50 mM KC1, 10 mM MgC12, 0.1 mM EDTA, 0.5 mM DTT, and

30 /xg/ml bovine serum albumin (BSA) for 6 hr at 29 ° in a reaction volume of 150 /xl 2° An amount of topoisomerase I sufficient to cause complete relaxation of the D N A templates is used Ethidium bromide and the topoisomerase are removed by two phenol extractions, one phenol : chloroform (1 : 1, v/v) extraction, and two chloroform extractions The topoisomers are ethanol precipitated and stored at - 8 0 ° Prior to phenol extraction, the fluorescence of each sample is measured on a PTI fluorimeter (Perkin-Elmer, Inc., Buckinghamshire, UK) to determine the amount of ethidium bromide bound to the template The average superhelical density of each sample is calculated according to the method of Singleton and Wells 2° and is verified by electrophoresis on a 1% (w/v) agarose gel in 1 x TBE (45 mM Tris-borate, 1 mM EDTA) containing appropriate concentrations of chloroquine

19K Backman, M J O'Connor, A Maruya, and M Erfle, Bio/Technology December,

1045 (1984)

2o C K Singleton and R D Wells, Anal Biochem 122, 253 (1982)

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14 BASAL TRANSCRIPTION AND REGULATION OF TRANSCRIPTION [2]

Probing Structure o f N4 Early Promoters on Supercoiled Templates with Chloroacetaldehyde, Mung Bean Nuclease, and T7 Endonuclease I

Chloroacetaldehyde (CAA) reactions are carried out as described previously, 21 with some modifications Reaction mixtures contain 10 m M Tris- HC1, pH 8.0, 50 m M NaC1, 1 m M EDTA, 10 m M MgC12, 1/~g of DNA, and 5% CAA in a total volume of 20 /~1 Mg 2+ is essential for hairpin extrusion; the cation specificity for hairpin extrusion suggests that Mg 2+ binds to and stabilizes the cruciform four-way junction 22'23 The components are gently mixed and incubated at 37 ° for 30 min Reactions are terminated

by the addition of 0.6 t~l of 5 M NaC1 and 50/.d of cold ethanol After precipitation, the sample is resuspended in 20/~1 of buffer containing 10

m M Tris-HC1, pH 8.0, 1 mM EDTA, and 150 m M NaCI and is reprecipi- tated with ethanol The pellet is then resuspended in 20/~1 of doubly distilled H20 and subjected to primer extension or piperidine cleavage analysis Mung bean nuclease cleavage reactions 24 are carried out for 30 rain at

37 ° in 10 m M Tris-HCl, pH 7.0, 50 mM NaC1, 0.5/~g DNA, and 10 m M MgCI2 in a final volume of 10/~1 The template is preincubated with the MgClz-containing buffer for 10 min at 37 ° prior to nuclease addition The amount of nuclease used is determined empirically by titration on different

D N A templates The reactions are terminated by phenol extraction, followed by ethanol precipitation The final products are resuspended in 10 /~1 of doubly distilled H20 and subjected to primer extension analysis Cleavage with T7 endonuclease I [generously provided by Dr W Studier, Brookhaven National Laboratory, as fraction 8 of a carboxymethyl (CM)-cellulose column 15] is carried out in 50 m M Tris-HC1, pH 8.0, 10

m M MgSO4, 1 mM DTT, 50/~g/ml BSA, 0.5/~g DNA, and T7 endonuclease

I for 30 min at 37 ° in a final volume of 50 IA The amount of nuclease used

is determined by titration experiments The reactions are terminated by phenol extraction, followed by ethanol precipitation The final products are resuspended in 10/zl of doubly distilled H20 and subjected to primer extension analysis

Mapping Modification~Cleavage Sites by Primer Extension Analys&

Primers that hybridize specifically to template and nontemplate sequences 40-60 bases away from each promoter are used to detect modifications or cleavages at the N4 early promoters by polymerase chain reaction

21 T Kohwi-Shigematsu, T Manes, and Y Kohwi, Proc Natl Acad Sci U.S.A 84, 2223 (1987) 2z j p Cooper and P J Hagerman, Proc Natl Acad Sci U.S.A 86, 7336 (1989)

23 D R Duckett, A I H Murchie, and D M Lilley, E M B O J 9, 583 (1990)

24 D Kowalski and J P Sanford, J Biol Chem 257, 7820 (1982)

Trang 36

[21 SINGLE-STRANDED DNA-BINDING PROTEINS 15 (PCR) amplification Approximately 0.01 pmol of modified or cleaved D N A

is mixed with 0.6 pmol of a 32p-labeled primer in Vent D N A polymerase buffer [10 m M KC1, 10 m M (NH4)2SO4, 20 mM Tris-HC1, pH 8.8, 2 m M MgSO4, 0.1% Triton X-100 (v/v)] containing 250 tzM of each dNTP in a total volume of 15/zl In most cases, 2 m M MgSO4 is sufficient to achieve optimal primer extension, although adjustments are sometimes necessary for specific template-primer pairs Two units of Vent (exo-) D N A polymer° ase (New England Biolabs, Beverly, MA) are added and the mixture is overlaid with one drop of mineral oil Twenty PCR cycles are performed using the following program: 20 sec at 95 °, 20 sec at the melting temperature

of the specific primer used, and 20 sec at 72 ° After cycling is completed,

9 tzl of loading buffer is added directly to the tubes The sample/dye mixture (3/zl) is loaded onto 6 or 8% polyacrylamide/7 M urea gels, alongside the products of double-stranded sequencing reactions performed on unmodi- fied D N A using the same primer and [35S]dATP (US Biochemicals Seque- nase protocol) We have confirmed that Vent polymerase stops elongation

at the site of C A A modification by end-labeling of the CAA-modified fragments at a unique site, followed by piperidine (10%) cleavage at 90 ° for 30 min These reaction mixtures are run on 8% polyacrylamide/7 M urea gels alongside Maxam-Gilbert sequencing ladders of the same fragmentY

Probing EcoSSB Interactions with N4 Promoters on

Single-Stranded Templates

DNase I footprinting is performed as described by Hoess and Abre- mski 26 The single-stranded D N A fragments are isolated by restricting the

M13mp7 viral strand containing N4 early promoters with BamHI or EcoRI

endonucleases TM The insert is 5' end-labeled by T4 polynucleotide kinase Each footprinting reaction includes 30 ng of the labeled fragment in DNase

I buffer (50 m M Tris-HCl, pH 7.5, 30 mM NaC1, 50/zg/ml BSA, 2 mM MgCI2, 1 m M DTT, 5% (v/v) glycerol) The DNase I cleavage reactions are carried out in a volume of 100/zl in the absence or presence of 30 ng

of EcoSSB (US Biochemicals) Protein and D N A are preincubated for 15

min at 37 °, transferred to 30 °, and treated with RQ1 DNase I (Promega) for 2 min The concentration of DNase I used is determined by titration The reactions are terminated by phenol extraction, followed by ethanol precipitation The pellets are resuspended in 4/zl doubly distilled H20 and 4/zl loading buffer, boiled for 3 min, and loaded onto 8% polyacrylamide/

7 M urea gels in 1× TBE buffer, alongside Maxam-Gilbert sequencing ladders 25 generated from the D N A template being footprinted

25 A M Maxam and W Gilbert, Methods Enzymol 65, 499 (1980)

26 R H Hoess and K Ambreski, Proc Natl Acad Sci U.S.A 81, 1026 (1984)

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16 B A S A L T R A N S C R I P T I O N A N D R E G U L A T I O N OF T R A N S C R I P T I O N [2]

Neocarzinostatin footprinting reaction mixtures 27 (100 tzl) contain 50

mM Tris-HC1, pH 7.5, 30 m M NaC1, 30 m M 2-mercaptoethanol, 10 mM MgC12, 50/zg/ml BSA, and 5% glycerol EcoSSB (30 ng) and D N A (30 ng) are incubated for 15 min at 37 ° and are treated with 10/zg neocarzinostatin (generously provided by Dr I Goldberg, Harvard Medical School, Cam- bridge, MA) for 10 min at room temperature The reactions are terminated by phenol extraction and processed as for DNase I footprinting reactions

Neurospora crassa nuclease footprinting reaction mixtures contain 10

m M Tris-HC1, pH 8.0, 10 m M MgC12, 50 m M NaC1, 1 m M EDTA, and

1 m M DTT 28 E c o S S B (30 ng) and D N A (30 ng) are incubated for 15 min

at 37 ° and are treated with 0.5 units of N crassa nuclease (Pharmacia) for

2 min at 30 ° The reaction is terminated with phenol and processed as described earlier

Role of N 4 S S B in Late T r a n s c r i p t i o n

Early genetic studies showed that the E coli o-7°-RNA polymerase

h o l o e n z y m e is responsible for N4 late transcription, with late transcripts appearing about 12 rain after infection 7 Late transcription is not dependent on N4 D N A replication, although N4 late transcripts appear after N4

D N A replication has started 7 The delay in N4 late transcription is due to the delayed production of a phage-encoded transcriptional activator, the single-stranded DNA-binding protein (N4SSB) 9 R u n o f f transcription assays using purified N4SSB 29 and E coli h o l o e n z y m e showed that N4SSB activates transcription from N4 late promoters resident on a linear template 9

T h e N4SSB (265 amino acids) gene was cloned into a tightly regulated, TT-directed overexpression vector for both protein purification and genetic studies 3° (see below) Mutational and biochemical analyses have led to a preliminary model of the functional domains of N4SSB 31 T h e C-terminal domain (consisting of approximately 10 residues) is involved in p r o t e i n - protein interactions T h e N-terminal domain contains the determinants of single-stranded D N A binding 31 These domains are separated by a small, flexible linker region

27 N L Craig and H A Nash, Cell 39, 707 (1984)

28 M J Fraser, Methods Enzymol 65, 255 (1980)

29 G J Lindberg, S C Kowalczykowski, J K Rist, A Sugino, and L B Rothman-Denes,

J BioL Chem 264, 12700 (1989)

3 0 M Choi, A Miller, N.-Y Cho, and L B Rothman-Denes, J Biol Chem 270, 22541 (1995)

31 A Miller, D Wood, M Choi, and L B Rothman-Denes, unpublished (1996)

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[2] SINGLE-STRANDED D N A - B I N D I N G PROTEINS 17

N4SSB does not bind to, nor can it denature natural double-stranded DNAs How, then, does it activate transcription? We have isolated mutants that are deficient in single-stranded D N A binding, but activate transcription both in vivo and in vitro, 32 indicating that the single-stranded DNA-binding activity is not required for transcriptional activation Furthermore, we have isolated mutations in the C-terminal domain that cause defects only in transcriptional activation 32 These results suggest that N4SSB, unlike proto- typical transcriptional activators, activates transcription at N4 late promoters by direct interaction with E coli R N A polymerase in the absence of specific D N A binding Three lines of evidence support this model: (1) Wild- type N4SSB coelutes with hexahistidine-tagged R N A polymerase 33 from Ni2+-NTA agarose affinity columns whereas activation-deficient N4SSB does not 32 (2) Biochemical and genetic data indicate that N4SSB contacts the C-terminal domain of the/3' subunit 32 (3) Because N4SSB is expressed

at very high levels during N4 infection (about 10,000 molecules per ce1129),

a DNA-binding site at the promoter might not be required to increase the local concentration of N4SSB for transcriptional activation At this point, the sequence requirements for N4SSB activation at the N4 late promoters and the transcription initiation step, which is affected by N4SSB, are not known

Methods

Cloning of N4SSB

Initial attempts to overproduce N4SSB using the T7 expression system 3° resulted in polypeptides smaller than N4SSB, indicating that its expression

is lethal to E coli Successful cloning required a tightly regulated system

in which the N4SSB gene was cloned under the direction of a T7 R N A polymerase promoter controlled by the lac operator34; the host strain carried the pcnB mutation, which decreases the copy number of pBR322 deriva- tives35; and the host contained the plasmid pLysE, 36 which supplies the T7 lysozyme, an inhibitor of T7 R N A polymerase 37 Plasmid pMC6, a pBR322 derivative, contains a 900-bp D N A fragment that includes a synthetic Shine-Dalgarno (SD) sequence, the N4SSB ORF, and the distal 100 non-

32 A Miller, D Wood, and L B R o t h m a n - D e n e s , submitted (1996)

33 D Wang, T I Meier, C L Chan, G Feng, D N Lee, and R Landick, Cell 81, 341 (1995)

34 W Studier, A H Rosenberg, J J Dunn, and J W Dubendorff, Methods EnzymoL 185,

60 (1990)

35 j W D u b e n d o r f f and F W Studier, J Mol BioL 219, 45 (1991)

36 j Lopilato, S Bortner, and J Beckwith, Mol Gen Genet 205, 285 (1986)

37 B m Moffatt and F W Studier, Cell 49, 221 (1987)

Trang 39

18 BASAL TRANSCRIPTION AND REGULATION OF TRANSCRIPTION [~] coding bases, all under the control of the T7 minimal promoter and the

lac operator from pET-11 34 Plasmid pMC6 is stable in BL21(DE3)/pLysE

in the absence of pcnB The production of cloned N4SSB protein is very efficient in this background 3°

Purification o f N4SSB

BL21(DE3)/pLysE carrying the N4SSB expression plasmid pMC6 is grown in LB media containing 0.1 mg/ml ampicillin and 50 tzg/ml chloram- phenicol to an OD620 of 0.5 IPTG (1 mM) is added and, after incubation for 100 min, ceils are collected and lysed in 10 mM Tris-HC1, pH 7.6, 20 mM EGTA, 0.2 M NaCI, 0.8 mg/ml lysozyme, and 0.5 mM phenylmethylsulfonyl fluoride After two cycles of freezing on dry ice-ethanol and thawing to

4 °, the lysate is centrifuged at 40,000 rpm for 1 hr at 4 ° To remove contami- nating polynucleotides, the lysate is treated by dropwise addition of 10% (w/v) streptomycin sulfate to a final concentration of 1.2% on ice, and the pellet is discarded after centrifugation at 10,000 rpm for 30 min Proteins precipitating between an ammonium sulfate concentration equal to 35 and 60% of saturation are resuspended in doubly distilled H20 and are applied

to a Superdex 75 (Pharmacia) 6.2 x 60-cm column equilibrated in buffer

A (50 mM Tris-HCl, pH 8, 10% glycerol (v/v), 1 mM EDTA, 5 mM 2- mercaptoethanol, 40 mM NaC1) at 4 ° At a flow rate of 1.5 ml/min of buffer

A, N4SSB elutes in the flow through of the column due to its ability to aggregate at high concentrations Samples (50/zl) from each fraction are TCA precipitated and analyzed by electrophoresis on a 12.5% SDS-PAGE gel N4SSB-containing fractions are loaded onto a 1 x 5-cm single-stranded DNA agarose column (Pharmacia) equilibrated with buffer A The column

is washed with 1 column volume of 1 mg/ml heparin in buffer A followed

by 3 column volumes of buffer A A bilinear gradient of 5 column volumes

of a 40 m M to 0.9 M NaC1 in buffer A and 5 column volumes of a 0.9 to

3 M NaCI in buffer A is applied to the column Fractions are analyzed by SDS-PAGE, and those containing 99% pure N4SSB are pooled and dialyzed against storage buffer containing 50 mM Tris-HC1, pH 8, 0.1 M NaC1, 20% glycerol, 1 mM EDTA, and 1 mM DTT and are frozen at - 8 0 ° N4SSB elutes at 1.5 M NaCI with a yield of 20 mg of protein from 3 liters of culture

In Vitro Runoff Transcription Assays

Linear DNA fragments bearing the N4 late promoter R are generated

by restriction of pUC(R) with BamHI 9'38 The 800-bp promoter-containing

~8 C Malone, S Spellman, D Hyman, and L B Rothman-Denes, Virology 162, 328 (1988)

Trang 40

[2] SINGLE-STRANDED DNA-BINDING PROTEINS 19 fragment is purified from 2% agarose gels and used as a template for in

10 rain at 37 °, the reaction is terminated by the addition of 2/xg of tRNA and E D T A to a final concentration of 25 raM The samples are extracted with phenol and the nucleic acids are precipitated with ethanol, resuspended

in loading buffer, and analyzed by electrophoresis on 8 M urea-8% polyacrylamide gels

Concluding Remarks

We have described two single-stranded DNA-binding proteins that act

as transcriptional activators through very different mechanisms Our results indicate that E c o S S B is a transcriptional activator by acting as an "architec- tural" protein, 39 i.e., by providing the correct D N A topology to the transcriptional machinery In this context, E c o S S B resembles integration host factor (IHF) 4° and the eukaryotic R N A polymerase I activator U B F 41

These proteins bind in a sequence-specific manner to D N A and bend the

D N A upon binding, allowing the contact of transcriptional activators with the respective transcriptional machineries Even though E c o S S B is not a sequence-specific DNA-binding protein, the unusual properties of the D N A

at the N4 virion R N A polymerase promoters provide specificity to the process of transcriptional activation

In contrast, the single-stranded DNA-binding activity of N4SSB is not required for transcriptional activation N4SSB is a multifunctional protein that contains a transcriptional activation domain responsible for direct interaction with E coli R N A polymerase 32 Why does bacteriophage N4 use its single-stranded DNA-binding protein for transcriptional activation?

In the context of viral development, it coordinates late gene expression with viral D N A replication 42

39 A P Wolfe, Science 264, 1100 (1994)

40 T R Hoover, E Santero, S Porter, and S Kustu, Cell 63, 11 (1990)

41 D Bazett-Jones, B Leblanc, M Herfort, and T Moss, Science 264, 1134 (1994)

42 E P Geiduschek, Semin ViroL 6, 25 (1995)

Tiêu đề	RNA Polymerase and Associated Factors, Part B
Tác giả	Sankar Adhya
Trường học	Pennsylvania State University
Chuyên ngành	Biochemistry and Molecular Biology
Thể loại	Lecture notes
Năm xuất bản	Not specified
Thành phố	University Park

Định dạng
Số trang	605
Dung lượng	10,73 MB