rna polymerase and associated factors, part d

Adamson 19, Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242 Michael Anikin 9, Morse Institute of Molecular Genetics, Department of Microbiology and Immunology, SUNY

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METHODS IN ENZYMOLOGY

EDITORS-IN-CHIEF

DIVISION OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY

PASADENA, CALIFORNIA

FOUNDING EDITORS

Sidney P Colowick and Nathan O Kaplan

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Affiliations listed are current.

Todd E Adamson (19), Department of

Biochemistry, University of Iowa, Iowa

City, Iowa 52242

Michael Anikin (9), Morse Institute of

Molecular Genetics, Department of

Microbiology and Immunology, SUNY

Downstate Medical Center, 450 Clarkson

Avenue, Brooklyn, New York 11203-2098

Ekaterina Avetissova (13), Skirball

Institute, New York University Medical

Center, New York, New York 10016

Paul Babitzke (30), Department of

Bio-chemistry and Molecular Biology, The

Pennsylvania State University, University

Park, Pennsylvania 16802

Gil Bar-Nahum (11), Department of

Bio-chemistry, New York University Medical

Irina Bass (14), Institute of Molecular

Genetics, Russian Academy of Sciences,

Moscow, 123182, Russia

Jodi Becker (17), NCI Center for Cancer

Research, National Cancer Institute—

Frederick Cancer Research and

Develop-ment Center, Frederick, Maryland 21702

Oxana Bereshchenko (14), Public Health

Research Institute, 455 First Avenue, New

York, New York 10016

Philip C Bevilacqua (30), Department of

Chemistry,

ThePennsylvaniaStateUniver-sity, University Park, Pennsylvania 16802

Kishor K Bhakat (22), Sealy Center for

Molecular Science and the Department of

Human Biological Chemistry and

Genet-ics, University of Texas Medical Branch,

Galveston, Texas 77555

Yehudit Birger (39), Protein Section, Laboratory of Metabolism, Center for Cancer Research, National Cancer Insti- tute, National Institutes of Health, Bethesda, Maryland 20892-4255 Sergei Borukov (16), Department of Microbiology and Immunology, SUNY Health Science Center at Brooklyn, 450 Clarkson Avenue Brooklyn, New York 11203-2098

Zachary F Burton (18), Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824

Michael Bustin (39), Protein Section, Laboratory of Metabolism, Center Na- tional Cancer Institute, National Insti- tutes of Health, Bethesda, Maryland 20892-4255

Marian Carlson (45), Department of etics & Development and Institute of Cancer Research, Columbia University, New York, New York 10032

Gen-Michael J Carrozza (40), Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802-4500

Michael Cashel (44), Laboratory of lecular Genetics, NICHD, NIH Building

Mo-6, Room 3B314 Bethesda, Maryland 20892-2785

Hyon Choy (38), Genome Center for teropathogenic Bacteria, Department of Microbiology, Chonnam National Univer- sity Medical College, Dong-Gu, Hak-1- Kwang-Ju 501-746, South Korea

En-xi

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Adrienne Clements (41), The Wistar

In-stitute, Philadelphia, Pennsylvania 19104

Ronald Conaway (20), Stowers Institute

for Medical Research, Kansas City,

Mis-souri 64110; Department of Biochemistry

and Molecular Biology, Kansas

Univer-sity Medical Center, Kansas City, Kansas

66160

Joan Weliky Conaway (20), Stowers

In-stitute for Medical Research, Kansas

City, Missouri 64110; Department of

Bio-chemistry and Molecular Biology, Kansas

University Medical Center, Kansas City,

Kansas 66160

Ciara´n Condon (35), CNRS UPR9073,

Institute de Biologie Physico-Chimigue,

75005 Paris, France

Asis Das (33), University of Connecticut

Health Center, Farmington, Connecticut

06030-3205

Saul A Datwyler (6), Abbott

Laborator-ies, 100 Abbott Park Rd, Abbott Park,

Illinois 60064

Richard H Ebright (10), Waksman

Insti-tute, Howard Hughes Medical InstiInsti-tute,

Rutgers University, 190 Frelinghuysen

Road, Piscataway, New Jersey 08854

Yon W Ebright (10), Waksman Institute,

Howard Hughes Medical Institute,

Rut-gers University, 190 Frelinghuysen Road,

Piscataway, New Jersey 08854

Vitaly Epshtein (14), Institute of

Molecu-lar Genetics, Russian Academy of

Sci-ences, Moscow, 123182, Russia

Dorothy A Erie (5), Department of

Chemistry, CB 3290, Venable and Kenan

Laboratories, University of North

Caroli-na at Chapel Hill, Chapel Hill, North

Carolina 27599-3290

Peggy J Farnham (43), McArdle

Labora-tory for Cancer Research, University of

Wisconsin Medical School 1400

Univer-sity Avenue, Madison, Wisconsin

53706-1599

Jane Fellows (36), Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Hertford- shire, EN6 3LD United Kingdom

J Estelle Foster (5), Department of Chemistry, CB 3290, Venable and Kenan Laboratories, University of North Caroli-

na at ChapelHill, ChapelHill, North olina 27599-3290

Car-David I Friedman (32), Department of Cell and Structure Biology, University of Colorado School of Medicine, 4200 E Ninth Avenue, UCHSC Campus, Box B-111 Denver, Colorado 80262

Dmitry V Fyodorov (37), Section of lecular Biology, University of California, San Diego, La Jolla, California 92093- 0347

Mo-Michell E Garber (24), Regulatory ogy Laboratory, Salk Institute for Bio- logical Studies, 10010 North Torrey Pines Road La Jolla, California 92037-1099 Alex Goldfarb (14), Public Health Re- search Institute, 455 First Avenue, New York, New York 10016

Biol-Paul Gollnick (31), Department of logical Sciences, Hochstetter Hall 613, State University of New York Buffalo, New York 14260

Bio-Nathan Gomes (24), Regulatory Biology Laboratory Salk Institute for Biological Studies, 10010 North Torrey Pines Road,

La Jolla, California 92037-1099 Xue Q Gong (18), Department of Bio- chemistry and Molecular Biology, Mich- igan State University, East Lansing, Michigan 48824

Feng Gong (29), Department of Biological Sciences, Stanford University, Stanford, California 94305-5020

Max E Gottesman (26), Institute of Cancer Research, Department of Bio- chemistry and Molecular Biophysics, Col- umbia University, New York, New York 10032-2798

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tute, Russian Academy of Sciences,

Ulan-batorskaya 3 664033, Russia

Ivan Gusarov (11, 28), Department of

Bio-chemistry, New York University Medical

Martin Guthold (3), Department of

Phys-ics, Wake Forest University

Winston-Salem, North Carolina 27109-7507

Hiroshi Handa (18), Frontier

Collabora-tive Research Center, Tokyo Institute of

Technology, Yokohama 226-8503, Japan

Ahmed H Hassan (40), Howard Hughes

Medical Institute, Department of

Park, Pennsylvania

Shannon F Holmes (4), Department of

Chemistry, CB 3290, Venable and Kenan

Laboratories, University of North

Caroli-na at Chapel Hill, Chapel Hill, North

Carolina 27599-3290

Masayori Inouye (34), Department of

Bio-chemistry, Robert Wood Johnson Medical

School, Piscataway, New Jersey

08854-5635

Akira Ishihama (6), Nippon Institute for

Biological Science, Shin-machi, 9-221

Ome, Tokyo 198-09924, Japan

Nandan Jana (33), University of

cut Health Center, Farmington,

Connecti-cut 06030-3205

Manli Jiang (9), Morse Institute of

Mo-lecular Genetics, Department of

Micro-biology and Immunology, SUNY

Downstate Medical Center 450 Clarkson

Avenue, Brooklyn, New York 11203-2098

Katherine A Jones (24), Regulatory

Biol-ogy Laboratory, Salk Institute for

Bio-logical Studies, 10010 North Torrey

Pines Road, La Jolla, California

92037-1099

lar Biology, University of California, San Diego, La Jolla, California 92093-0347 Changwon Kang (12), Department of Bio- logical Sciences, Korea Advanced Insti- tute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 701, Republic of Korea

Achillefs N Kapanidis (10), Department

of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095

Valeri N Karamychev (7), Department of Nuclear Medicine, Warren G Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892 Mikhail Kashlev (7, 14, 17, 42), NCI Center for Cancer Research, National Cancer Institute—Frederick Cancer Re- search and Development Center, Fred- erick, Maryland 21702

Hyeong C Kim (26), Department of chemistry and Molecular Biophysics Col- umbia University New York, New York 10032-2798

Bio-Jae-Hong Kim (38), Aging and Apoptosis, Research Center, Dept of Biochemistry and Molecular Biology, College of Medi- cine, Seoul National University, 28 Yon- gon-Dong, Chongno-GU, Seoul 110-799, South Korea

Rodney A King (15), Laboratory of lecular Genetics, NICHD, NIH, Building 6B, Room 3B-308, Bethesda, Maryland 20892

Mo-Maria L Kireeva (17, 42), NCI Center for Cancer Research, National Cancer Insti- tute—Frederick Cancer Research Devel- opment Center, Frederick, Maryland 21702

Natalia Komissarova (7, 17), NCI Center for Cancer Research National Cancer In- stitute—Frederick Cancer Research and Development Center, Frederick, Mary- land 21702

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Stephanie Kong (20), Stowers Institute

Medical Research, 1000 E 50th Street,

Kansas City, Missouri 64110

Ekaterine Kortkhonjia (10), Waksman

Institute, Howard Hughes Medical

Insti-tute, Rutgers University, 190

Frelinghuy-sen Road, Piscataway, New Jersey 08854

Nataliya Korzheva (13, 14), Public

Health Research Institute, 225 Warren

Street, Newark, New Jersey 07103-3535

Maxim Kozlov (14), Public Health

Re-search Institute, 455 First Avenue, New

York, New York 10016

Sergei Kuchin (45) Institute of Cancer

Re-search, Columbia University, New York,

New York 10032

Oleg Laptenko (16), Morse Institute of

Molecular Genetics, Department of

Microbiology and Immunology, SUNY

Health Science Center at Brooklyn, 450

Clarkson Avenue, Brooklyn, New York

11203-2098

David Lazinski (33), University of

necticut Health Center, Farmington,

Con-necticut 06030-3205

Lasse Lindahl (27), Department of

Bio-logical Sciences, University of Maryland,

Baltimore Country, 1000 Hilltop Circle,

Baltimore, Maryland 21250

Cuihua Liu (2), Wyeth Research, 87

Cam-bridgepark Drive, Cambridge,

Massachu-setts 02140

Eugeny Lukhtanov (14), Epoch

Pharma-ceuticals, Inc., Bothell, Washington 98021

Kaiyu Ma (9), Morse Institute of Molecular

Genetics, Department of Microbiology and

Immunology, SUNY Downstate Medical

Center, Brooklyn, New York 11203-2098

Vadim Markovtsov (14), Public Health

Research Institute, 455 First Avenue,

New York, New York 10016

Ronen Marmorstein (41), The Wistar

In-stitute, Philadelphia, Pennsylvania 19104

Craig T Martin (2), Department of istry, University of Massachusetts, Am- herst, Massachusetts 01003-9336 Tatyana Maximova (14), Limnological In- stitute, Russian Academy of Sciences, Ulanbatorskaya 3 664033, Russia William T McAllister (9), Morse Insti- tute of Molecular Genetics, Department

Chem-of Microbiology and Immunology, SUNY Downstate Medical Center 450 Clarkson Avenue Brooklyn, New York 11203-2098 Claude F Meares (6), Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, Cali- fornia 95616-5295

Vladimir Mekler (10), Waksman tute, Howard Hughes Medical Institute, Rutgers University, 190 Frelinghuysen Road, Piscataway, New Jersey 08854 Jaime Garcia Mena (33), University of Connecticut Health Center, Farmington, Connecticut 06030-3205

Insti-Gregory Michaud (33), University of Connecticut Health Center, Farmington, Connecticut 06030-3205

Sankar Mitra (22), Sealy Center for lecular Science, University of Texas, Medical Branch, 6.136 Medical Research Building, Galveston, Texas 77555 Mark Mortin (46), Laboratory of Molec- ular Genetics NICHD, NIH, Building 6B, Room 3B-331, Bethesda, Maryland 20892-4255

Mo-Jayanta Mukhopadhyay (10), Waksman Institute, Howard Hughes Medical Insti- tute, Rutgers University, 190 Frelinghuy- sen, Road, Piscataway, New Jersey 08854 Helen Murphy (44), Laboratory of Mo- lecular Genetics, NICHD, NIH Building

6, Room 3B-314, Bethesda, Maryland 20892-2785

Arkady Mustaev (13, 14), Public Health Research Institute, 455 First Avenue, New York, New York 10016

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Biochemistry, Michigan State University,

East Lansing, Michigan 48824

Melody N Neeley (32), Department of

Microbiology and Immunology, The

Uni-versity of Michigan Medical School, Ann

Arbor, Michigan 48109-0620

Ronald D Neumann (7), Department of

Nuclear Medicine, Warren G Magnuson

Clinical Center, National Institutes of

Health, Bethesda, Maryland 20892

Vadim Nikiforov (14), Institute of

Molecu-lar Genetics, Russian Academy of

Sci-ences, Moscow, 123182, Russia

Evgeny Nudler (11, 13, 28), Department of

Biochemistry, New York University

Med-ical Center, New York, New York 10016

Matthew J Oberley (43), Department of

Oncology, Room 417A McArdle

Labora-tory of Cancer Research, University of

Wisconsin-Madison, 1400 University

Avenue, Madison, Wisconsin 53706-1599

Jeffrey Owens (6), IDEC

Pharmaceut-icals, 3031 Science Park Road, San Diego,

CA 92191

Igor G Pantyutin (7), Department of

Nu-clear Medicine, Warren G Magnuson

Clinical Center, National Institutes of

Health, Bethesda, Maryland 20892

Sang Chul Park (38), Aging and

Apop-tosis Research Center, Department of

Biochemistry and Molecular Biology,

College of Medicine, Seoul National

Uni-versity, 28 Yongon-Dong Chongno-GU,

Seoul 110-799, South Korea

Thodoris G Petrakis (36), Cancer

Re-search UK, London ReRe-search Institute,

Clare Hall Laboratories, South Mimms,

Hertfordshire EN6 3LD, United Kingdom

Sangita Phadtare (34), Department of

Biochemistry, Robert Wood Johnson

Medical School, 675 Hoes Lane

Piscat-away, New Jersey 08854-5635

Laboratory of Metabolism, Center for Cancer Research, National Cancer Insti- tute, National Institutes of Health, Bethesda, Maryland 20892-4255 David H Price (19), Department of Bio- chemistry, University of Iowa, Iowa City, Iowa 52242

Daniel Reines (21), Department of chemistry, Emory University School of Medicine, 4023 Rollins Research Center, Atlanta, Georgia 30322

Claudio Rivetti (3), Dipartimento di chimica e Biologia, Molecolare Universita degli Studi, di Parma Parco Area, delle Scienze 23/A 43100, Parma, Italy Jeffrey W Roberts (8), Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853 Aziz Sancar (23), Department of Biochem- istry and Biophysics, University of North Carolina, School of Medicine, Chapel Hill, North Carolina 27599-7260 Thomas J Santangelo (8), Department

Bio-of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853

Janell Schaak (30), Department of chemistry and Molecular Biology and De- partment of Chemistry, The Pennsylvania State University, University Park, Penn- sylvania 16802

Bio-Brian D Schmidt (6), Onyx icals, 3031 Research Drive, Richmond, California 94806

Pharmaceut-C P Selby (23), Department of try and Biophysics, University of North Carolina, School of Medicine, Chapel Hill, North Carolina 27599-7260 Ranjan Sen (15), Laboratory of Molecular Genetics, NICHD, NIH, Bldg 6B, Rm 3B-308, Bethesda, Maryland 20892

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Biochemis-Sibani Sengupta (33), University of

Con-necticut Health Center, 263 Farmington

Avenue, Farmington, Connecticut

06030-3205

Hyuk-Kyu Seoh (35), Department of

Mo-lecular Biology and Microbiology, Tufts

University School of Medicine, Boston,

Massachusetts 02111-1800

Konstantin Severinov (14, 34), Waksman

Institute of Microbiology, Rutgers, The

State University of New Jersey, 190

Fre-linghuysen Road, Piscataway, New Jersey

08854

Haihong Shen (12), Department of

Bio-logical Sciences, Korea Advanced

Insti-tute of Science and Technology, 373-1

Guesong-dong, Yuseong-gu, Daejeon

305-701 Korea

Ali Shilatifard (20), Edward A Doisey,

Department of Biochemistry, St Louis

University School of Medicine, St Louis,

Missouri 63104

Nubuo Shimamoto (4), National

Institute of Genetics, School of Life

Science, The Graduate University for

Advanced Studies, Mishima, 411-8540

Japan

Sarah M Shore (19), Department of

Bio-chemistry, University of Iowa, Iowa City,

Iowa 52242

Igor Sidorenkov (17), NCI Center for

Cancer Research, National Cancer

Insti-tute—Frederick, Cancer Research

Devel-opment Center, Frederick, Maryland

21702

Younghee Sohn (12), Department of

Bio-logical Sciences, Korea Advanced

Insti-tute of Science and Technology, 373-1

Guseong-dong Yuseong-gu, Daejeon

305-701, Republic of Korea

Rui Sousa (1), Department of

Biochemis-try, University of Texas Health Science

Center, 7703 Floyd Court Drive, San

An-tonio, Texas 78284-7760

Catherine L Squires (35), Department

of Molecular Biology and Microbiology, Tufts University, School of Medicine, Boston, Massachusetts 02111-1800 Vasily M Studitsky (42), Department of Biochemistry and Molecular Biology, Wayne State University School of Medi- cine, Detroit, Michigan 48201

Jesper Q Svejstrup (36), Cancer Research

UK, London Research Institute, Clare Hall Laboratories, South Mimms, Hert- fordshire EN6 3LD, United Kingdom Alexi Tatusov (7), Department of Nuclear Medicine, Warren G Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892

Vladimir Tchernajenko (566), ment of Biochemistry and Molecular Biol- ogy, Wayne State University School of Medicine, Detroit, Michigan 48201 Dmitri Temiakov (9), Morse Institute of Molecular Genetics, Department of Microbiology and Immunology, SUNY Downstate 450 Clarkson Avenue, Brook- lyn, New York 11203-2098

Depart-Igor Tsarev (14), Limnological Institute, Russian Academy of Sciences, Ulanba- torskaya 3 664033, Russia

Andrea U ´ jva´ri (2), Department of lecular Biology, Lerner Research Insti- tute, Cleveland Clinic Foundation, Cleveland, Ohio 44195

Susan Uptain (25), Department of lecular Genetics and Cell Biology, Univer- sity of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637

Mo-Wendy Walter (7), Department of chemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201

Bio-Robert A Weisberg (15), Laboratory of Molecular Genetics, NICHD, NIH Build- ing 6B, Room 3B-308, Bethesda, Maryland 20892

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Laboratory of Metabolism, Center for

Cancer Research, National Cancer

Insti-tute, National Institutes of Health,

Bethesda, Maryland 20892-4255

Jerry L Workman (40), Howard Hughes

Medical Institute, Department of

Park, Pennsylvania 16802-4500

Alexander V Yakhnin (30), Department

of Biochemistry and Molecular Biology,

The Pennsylvania State University,

Uni-versity Park, Pennsylvania 16802

Yuki Yamaguchi (18), Graduate School of

Bioscience and Biotechnology, Tokyo

In-stitute of Technology, Yokohama

226-8503, Japan

Suk Hoon Yang (22), Sealy Center for

Molecular Science and the Department

of Human Biological Chemistry and

Gen-etics, University of Texas Medical

Branch, Galveston, Texas 77555

sor of Biology, Department of Biological Sciences, Stanford University, Stanford, California 94305-5020

Eugeny Zaychikov (14), stitute for Biochemie 8033, Martinsreid bei, Munchen, Germany

Max-Planck-In-Janice M Zengel (27), Department of Biological Sciences, University of Mary- land Baltimore Country, Baltimore, Maryland 21250

Zuo Zhang (33), University of Connecticut Health Center, Farmington, Connecticut, 06030-3205

Victor Zhurkin (7), Laboratory of putational and Experimental Biology, Na- tional Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

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Com-METHODS IN ENZYMOLOGY

Volume I Preparation and Assay of Enzymes

Edited by Sidney P Colowick and Nathan O Kaplan

Volume II Preparation and Assay of Enzymes

Volume III Preparation and Assay of Substrates

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

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

xix

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Edited by Kenneth Kustin

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 Kivie 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 Timasheff

Volume XXVI Enzyme Structure (Part C)

Volume XXVII Enzyme Structure (Part D)

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)

Volume XXXI Biomembranes (Part A)

Edited by Sidney Fleischer and Lester Packer

Volume XXXII Biomembranes (Part B)

Volume XXXIII Cumulative Subject Index Volumes I-XXX

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

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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

Volume XXXVII Hormone Action (Part B: Peptide Hormones)

Edited by Bert W O’Malley and Joel G Hardman

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 OrganSystems)

Edited by Joel G Hardman and Bert W O’Malley

Volume XL Hormone Action (Part E: Nuclear Structure and Function)Edited by Bert W O’Malley and Joel G Hardman

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)

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Volume LV Biomembranes (Part F: Bioenergetics)

Volume LVI Biomembranes (Part G: Bioenergetics)

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)

Volume LX Nucleic Acids and Protein Synthesis (Part H)

Volume 61 Enzyme Structure (Part H)

Volume 62 Vitamins and Coenzymes (Part D)

Volume 63 Enzyme Kinetics and Mechanism (Part A: Initial Rate andInhibitor Methods)

Edited by Daniel L Purich

Volume 64 Enzyme Kinetics and Mechanism (Part B: Isotopic Probes andComplex 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)

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Volume 72 Lipids (Part D)

Volume 73 Immunochemical Techniques (Part B)

Edited by John J Langone and Helen Van Vunakis

Volume 74 Immunochemical Techniques (Part C)

Volume 75 Cumulative Subject Index Volumes XXXI, XXXII, XXXIV–LXEdited by Edward A Dennis and Martha G Dennis

Volume 76 Hemoglobins

Edited by Eraldo Antonini, Luigi Rossi-Bernardi, and Emilia ChianconeVolume 77 Detoxication and Drug Metabolism

Volume 78 Interferons (Part A)

Edited by Sidney Pestka

Volume 79 Interferons (Part B)

Volume 80 Proteolytic Enzymes (Part C)

Edited by Laszlo Lorand

Volume 81 Biomembranes (Part H: Visual Pigments and Purple Membranes, 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 ContractileApparatus 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 Membranes, II)Edited by Lester Packer

Volume 89 Carbohydrate Metabolism (Part D)

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Volume 91 Enzyme Structure (Part I)

Volume 92 Immunochemical Techniques (Part E: Monoclonal Antibodies andGeneral Immunoassay Methods)

Volume 93 Immunochemical Techniques (Part F: Conventional Antibodies,

Fc Receptors, and Cytotoxicity)

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 andTargeting (General Methods; Eukaryotes)]

Edited by Sidney Fleischer and Becca Fleischer

Volume 97 Biomembranes [Part K: Membrane Biogenesis: Assembly andTargeting (Prokaryotes, Mitochondria, and Chloroplasts)]

Volume 98 Biomembranes (Part L: Membrane Biogenesis: Processing andRecycling)

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 Wu, Lawrence Grossman, and Kivie Moldave

Volume 102 Hormone Action (Part G: Calmodulin and Calcium-BindingProteins)

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

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Volume 107 Posttranslational Modifications (Part B)

Edited by Finn Wold and Kivie Moldave

Volume 108 Immunochemical Techniques (Part G: Separation and

Characterization 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

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 Rilling

Volume 112 Drug and Enzyme Targeting (Part A)

Edited by Kenneth J Widder and Ralph Green

Volume 113 Glutamate, Glutamine, Glutathione, and Related CompoundsEdited by Alton Meister

Volume 114 Diffraction Methods for Biological Macromolecules (Part A)Edited by Harold W Wyckoff, C H W Hirs, and Serge N TimasheffVolume 115 Diffraction Methods for Biological Macromolecules (Part B)Edited by Harold W Wyckoff, C H W Hirs, and Serge N TimasheffVolume 116 Immunochemical Techniques (Part H: Effectors and Mediators ofLymphoid Cell Functions)

Edited by Giovanni Di Sabato, John J Langone, and Helen Van VunakisVolume 117 Enzyme Structure (Part J)

Volume 118 Plant Molecular Biology

Edited by Arthur Weissbach and Herbert Weissbach

Volume 119 Interferons (Part C)

Volume 121 Immunochemical Techniques (Part I: Hybridoma Technologyand 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)

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and Chloroplasts: General Approaches and Transport Systems)

Volume 126 Biomembranes (Part N: Transport in Bacteria, Mitochondria, andChloroplasts: Protonmotive Force)

Volume 127 Biomembranes (Part O: Protons and Water: Structure andTranslocation)

Volume 128 Plasma Lipoproteins (Part A: Preparation, Structure, andMolecular Biology)

Edited by Jere P Segrest and John J Albers

Volume 129 Plasma Lipoproteins (Part B: Characterization, Cell Biology, andMetabolism)

Edited by John J Albers and Jere P Segrest

Volume 130 Enzyme Structure (Part K)

Volume 131 Enzyme Structure (Part L)

Volume 132 Immunochemical Techniques (Part J: Phagocytosis and

Cell-Mediated 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 ContractileApparatus 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-BindingProteins)

Edited by Anthony R Means and P Michael Conn

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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

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 Sirbasku

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 TCell 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 DNA (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 RelatedTransport: The Na, K-Pump)

Volume 157 Biomembranes (Part Q: ATP-Driven Pumps and RelatedTransport: Calcium, Proton, and Potassium Pumps)

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Edited by James F Riordan and Bert L Vallee

Volume 159 Initiation and Termination of Cyclic Nucleotide ActionEdited 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 Inflammation)

Volume 163 Immunochemical Techniques (Part M: Chemotaxis

and Inflammation)

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)Edited by P Michael Conn

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

Edited by Jacek Hawiger

Volume 170 Nucleosomes

Edited by Paul M Wassarman and Roger D Kornberg

Volume 171 Biomembranes (Part R: Transport Theory: Cells and ModelMembranes)

Volume 172 Biomembranes (Part S: Transport: Membrane Isolation andCharacterization)

Volume 173 Biomembranes [Part T: Cellular and Subcellular Transport:Eukaryotic (Nonepithelial) Cells]

Volume 174 Biomembranes [Part U: Cellular and Subcellular Transport:Eukaryotic (Nonepithelial) Cells]

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Volume 176 Nuclear Magnetic Resonance (Part A: Spectral Techniques andDynamics)

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)

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

Volume 182 Guide to Protein Purification

Edited by Murray P Deutscher

Volume 183 Molecular Evolution: Computer Analysis of Protein and NucleicAcid 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 Radicalsand 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)Edited by Lester Packer

Volume 191 Biomembranes (Part V: Cellular and Subcellular Transport:Epithelial Cells)

Volume 192 Biomembranes (Part W: Cellular and Subcellular Transport:Epithelial Cells)

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Edited by James A McCloskey

Volume 194 Guide to Yeast Genetics and Molecular Biology

Edited by Christine Guthrie and Gerald R Fink

Volume 195 Adenylyl Cyclase, G Proteins, and Guanylyl CyclaseEdited by Roger A Johnson and Jackie D Corbin

Volume 196 Molecular Motors and the Cytoskeleton

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

Volume 199 Cumulative Subject Index Volumes 168–174, 176–194Volume 200 Protein Phosphorylation (Part A: Protein Kinases: Assays,Purification, Antibodies, Functional Analysis, Cloning, and Expression)Edited by Tony Hunter and Bartholomew M Sefton

Volume 201 Protein Phosphorylation (Part B: Analysis of ProteinPhosphorylation, 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 RelatedMolecules)

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 Protein–DNA Interactions

Edited by Robert T Sauer

Volume 209 Phospholipid Biosynthesis

Edited by Edward A Dennis and Dennis E Vance

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Volume 210 Numerical Computer Methods

Edited by Ludwig Brand and Michael L Johnson

Volume 211 DNA Structures (Part A: Synthesis and Physical Analysis ofDNA)

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, andAntioxidation)

Volume 214 Carotenoids (Part B: Metabolism, Genetics, and Biosynthesis)Edited by Lester Packer

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

Edited by Jacek J Hawiger

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 Du¨zgu¨nes,

Volume 221 Membrane Fusion Techniques (Part B)

Volume 222 Proteolytic Enzymes in Coagulation, Fibrinolysis, and

Complement Activation (Part A: Mammalian Blood Coagulation Factors andInhibitors)

Edited by Laszlo Lorand and Kenneth G Mann

Volume 223 Proteolytic Enzymes in Coagulation, Fibrinolysis, and

Complement Activation (Part B: Complement Activation, Fibrinolysis, andNonmammalian 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, andAllan C Wilson

Volume 225 Guide to Techniques in Mouse Development

Edited by Paul M Wassarman and Melvin L DePamphilis

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Methods for Probing Metal Ion Environments in Metalloenzymes andMetalloproteins)

Volume 227 Metallobiochemistry (Part D: Physical and SpectroscopicMethods for Probing Metal Ion Environments in Metalloproteins)

Volume 228 Aqueous Two-Phase Systems

Edited by Harry Walter and Go¨te 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 WinslowVolume 232 Hemoglobins (Part C: Biophysical Methods)

Edited by Johannes Everse, Kim D Vandegriff, and Robert M WinslowVolume 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 ofVirulence Factors)

Edited by Virginia L Clark and Patrik M Bavoil

Volume 236 Bacterial Pathogenesis (Part B: Integration of PathogenicBacteria with Host Cells)

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 Shafer

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

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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 Barrett

Volume 249 Enzyme Kinetics and Mechanism (Part D: Developments inEnzyme Dynamics)

Volume 250 Lipid Modifications of Proteins

Edited by Patrick J Casey and Janice E Buss

Volume 251 Biothiols (Part A: Monothiols and Dithiols, Protein Thiols, andThiyl Radicals)

Volume 252 Biothiols (Part B: Glutathione and Thioredoxin; Thiols in SignalTransduction 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)Edited by W E Balch, Channing J Der, and Alan Hall

Volume 257 Small GTPases and Their Regulators (Part C: Proteins Involved

in Transport)

Edited by W E Balch, Channing J Der, and Alan Hall

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

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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 AnalysisEdited by Russell F Doolittle

Volume 267 Combinatorial Chemistry

Edited by John N Abelson

Volume 268 Nitric Oxide (Part A: Sources and Detection of NO; NOSynthase)

Volume 269 Nitric Oxide (Part B: Physiological and

Pathological Processes)

Volume 270 High Resolution Separation and Analysis of BiologicalMacromolecules (Part A: Fundamentals)

Edited by Barry L Karger and William S Hancock

Volume 271 High Resolution Separation and Analysis of BiologicalMacromolecules (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 Steven S CarrollVolume 276 Macromolecular Crystallography (Part A)

Edited by Charles W Carter, Jr., and Robert M Sweet

Volume 277 Macromolecular Crystallography (Part B)

Volume 278 Fluorescence Spectroscopy

Edited by Ludwig Brand and Michael L Johnson

Volume 279 Vitamins and Coenzymes (Part I)

Edited by Donald B McCormick, John W Suttie, and Conrad Wagner

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Volume 280 Vitamins and Coenzymes (Part J)

Edited by Donald B McCormick, John W Suttie, and Conrad WagnerVolume 281 Vitamins and Coenzymes (Part K)

Edited by Donald B McCormick, John W Suttie, and Conrad WagnerVolume 282 Vitamins and Coenzymes (Part L)

Edited by Donald B McCormick, John W Suttie, and Conrad WagnerVolume 283 Cell Cycle Control

Edited by William G Dunphy

Volume 284 Lipases (Part A: Biotechnology)

Edited by Byron Rubin and Edward A Dennis

Volume 285 Cumulative Subject Index Volumes 263, 264, 266–284, 286–289Volume 286 Lipases (Part B: Enzyme Characterization and Utilization)Edited by Byron Rubin and Edward A Dennis

Volume 287 Chemokines

Edited by Richard Horuk

Volume 288 Chemokine Receptors

Edited by Richard Horuk

Volume 289 Solid Phase Peptide Synthesis

Edited by Gregg B Fields

Volume 290 Molecular Chaperones

Edited by George H Lorimer and Thomas Baldwin

Volume 291 Caged Compounds

Edited by Gerard Marriott

Volume 292 ABC Transporters: Biochemical, Cellular, and Molecular AspectsEdited by Suresh V Ambudkar and Michael M Gottesman

Volume 293 Ion Channels (Part B)

Volume 294 Ion Channels (Part C)

Volume 295 Energetics of Biological Macromolecules (Part B)

Edited by Gary K Ackers and Michael L Johnson

Volume 296 Neurotransmitter Transporters

Edited by Susan G Amara

Volume 297 Photosynthesis: Molecular Biology of Energy Capture

Edited by Lee McIntosh

Volume 298 Molecular Motors and the Cytoskeleton (Part B)

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Volume 300 Oxidants and Antioxidants (Part B)

Volume 301 Nitric Oxide: Biological and Antioxidant Activities (Part C)Edited by Lester Packer

Volume 302 Green Fluorescent Protein

Volume 303 cDNA Preparation and Display

Edited by Sherman M Weissman

Volume 304 Chromatin

Edited by Paul M Wassarman and Alan P Wolffe

Volume 305 Bioluminescence and Chemiluminescence (Part C)

Edited by Thomas O Baldwin and Miriam M Ziegler

Volume 306 Expression of Recombinant Genes in Eukaryotic SystemsEdited by Joseph C Glorioso and Martin C Schmidt

Volume 307 Confocal Microscopy

Volume 308 Enzyme Kinetics and Mechanism (Part E: Energetics of EnzymeCatalysis)

Edited by Daniel L Purich and Vern L Schramm

Volume 309 Amyloid, Prions, and Other Protein Aggregates

Edited by Ronald Wetzel

Volume 310 Biofilms

Edited by Ron J Doyle

Volume 311 Sphingolipid Metabolism and Cell Signaling (Part A)

Edited by Alfred H Merrill, Jr., and Yusuf A Hannun

Volume 312 Sphingolipid Metabolism and Cell Signaling (Part B)

Edited by Alfred H Merrill, Jr., and Yusuf A Hannun

Volume 313 Antisense Technology (Part A: General Methods, Methods ofDelivery, and RNA Studies)

Edited by M Ian Phillips

Volume 314 Antisense Technology (Part B: Applications)

Volume 315 Vertebrate Phototransduction and the Visual Cycle (Part A)Edited by Krzysztof Palczewski

Volume 316 Vertebrate Phototransduction and the Visual Cycle (Part B)Edited by Krzysztof Palczewski

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Volume 317 RNA–Ligand Interactions (Part A: Structural Biology Methods)Edited by Daniel W Celander and John N Abelson

Volume 318 RNA–Ligand Interactions (Part B: Molecular Biology Methods)Edited by Daniel W Celander and John N Abelson

Volume 319 Singlet Oxygen, UV-A, and Ozone

Edited by Lester Packer and Helmut Sies

Volume 320 Cumulative Subject Index Volumes 290–319

Volume 321 Numerical Computer Methods (Part C)

Edited by Michael L Johnson and Ludwig Brand

Volume 322 Apoptosis

Edited by John C Reed

Volume 323 Energetics of Biological Macromolecules (Part C)

Edited by Michael L Johnson and Gary K Ackers

Volume 324 Branched-Chain Amino Acids (Part B)

Edited by Robert A Harris and John R Sokatch

Volume 325 Regulators and Effectors of Small GTPases (Part D: Rho Family)Edited by W E Balch, Channing J Der, and Alan Hall

Volume 326 Applications of Chimeric Genes and Hybrid Proteins (Part A:Gene Expression and Protein Purification)

Edited by Jeremy Thorner, Scott D Emr, and John N Abelson

Volume 327 Applications of Chimeric Genes and Hybrid Proteins (Part B:Cell Biology and Physiology)

Volume 328 Applications of Chimeric Genes and Hybrid Proteins (Part C:Protein–Protein Interactions and Genomics)

Volume 329 Regulators and Effectors of Small GTPases (Part E: GTPasesInvolved in Vesicular Traffic)

Edited by W E Balch, Channing J Der, and Alan Hall

Volume 330 Hyperthermophilic Enzymes (Part A)

Edited by Michael W W Adams and Robert M Kelly

Volume 331 Hyperthermophilic Enzymes (Part B)

Volume 332 Regulators and Effectors of Small GTPases (Part F: Ras Family I)Edited by W E Balch, Channing J Der, and Alan Hall

Volume 333 Regulators and Effectors of Small GTPases (Part G: Ras Family II)Edited by W E Balch, Channing J Der, and Alan Hall

Volume 334 Hyperthermophilic Enzymes (Part C)

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Volume 336 Microbial Growth in Biofilms (Part A: Developmental andMolecular Biological Aspects)

Volume 337 Microbial Growth in Biofilms (Part B: Special Environments andPhysicochemical Aspects)

Volume 338 Nuclear Magnetic Resonance of Biological Macromolecules(Part A)

Edited by Thomas L James, Volker Do¨tsch, and Uli Schmitz

Volume 339 Nuclear Magnetic Resonance of Biological Macromolecules(Part B)

Edited by Thomas L James, Volker Do¨tsch, and Uli Schmitz

Volume 340 Drug–Nucleic Acid Interactions

Edited by Jonathan B Chaires and Michael J Waring

Volume 341 Ribonucleases (Part A)

Edited by Allen W Nicholson

Volume 342 Ribonucleases (Part B)

Edited by Allen W Nicholson

Volume 343 G Protein Pathways (Part A: Receptors)

Edited by Ravi Iyengar and John D Hildebrandt

Volume 344 G Protein Pathways (Part B: G Proteins and Their Regulators)Edited by Ravi Iyengar and John D Hildebrandt

Volume 345 G Protein Pathways (Part C: Effector Mechanisms)

Edited by Ravi Iyengar and John D Hildebrandt

Volume 346 Gene Therapy Methods

Volume 347 Protein Sensors and Reactive Oxygen Species (Part A:

Selenoproteins and Thioredoxin)

Edited by Helmut Sies and Lester Packer

Volume 348 Protein Sensors and Reactive Oxygen Species (Part B: ThiolEnzymes and Proteins)

Edited by Helmut Sies and Lester Packer

Volume 349 Superoxide Dismutase

Volume 350 Guide to Yeast Genetics and Molecular and Cell Biology (Part B)Edited by Christine Guthrie and Gerald R Fink

Volume 351 Guide to Yeast Genetics and Molecular and Cell Biology (Part C)Edited by Christine Guthrie and Gerald R Fink

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Volume 352 Redox Cell Biology and Genetics (Part A)

Edited by Chandan K Sen and Lester Packer

Volume 353 Redox Cell Biology and Genetics (Part B)

Edited by Chandan K Sen and Lester Packer

Volume 354 Enzyme Kinetics and Mechanisms (Part F: Detection andCharacterization of Enzyme Reaction Intermediates)

Volume 355 Cumulative Subject Index Volumes 321–354

Volume 356 Laser Capture Microscopy and Microdissection

Volume 357 Cytochrome P450, Part C

Edited by Eric F Johnson and Michael R Waterman

Volume 358 Bacterial Pathogenesis (Part C: Identification, Regulation, andFunction of Virulence Factors)

Volume 359 Nitric Oxide (Part D)

Edited by Enrique Cadenas and Lester Packer

Volume 360 Biophotonics (Part A)

Edited by Gerard Marriott and Ian Parker

Volume 361 Biophotonics (Part B)

Edited by Gerard Marriott and Ian Parker

Volume 362 Recognition of Carbohydrates in Biological Systems (Part A)Edited by Yuan C Lee and Reiko T Lee

Volume 363 Recognition of Carbohydrates in Biological Systems (Part B)Edited by Yuan C Lee and Reiko T Lee

Volume 364 Nuclear Receptors

Edited by David W Russell and David J Mangelsdorf

Volume 365 Differentiation of Embryonic Stem Cells

Edited by Paul M Wassauman and Gordon M Keller

Volume 366 Protein Phosphatases

Edited by Susanne Klumpp and Josef Krieglstein

Volume 367 Liposomes (Part A)

Volume 368 Macromolecular Crystallography (Part C)

Volume 369 Combinational Chemistry (Part B)

Edited by Guillermo A Morales and Barry A Bunin

Volume 370 RNA Polymerases and Associated Factors (Part C)

Edited by Sankar L Adhya and Susan Garges

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Edited by Sankar L Adhya and Susan Garges

Volume 372 Liposomes (Part B)

Edited by Negat Du¨zgu¨nes,

Volume 373 Liposomes (Part C)

Edited by Negat Du¨zgu¨nes,

Volume 374 Macromolecular Crystallography (Part D)

Edited by Charles W Carter, Jr., and Robert W Sweet

Volume 375 Chromatin and Chromatin Remodeling Enzymes (Part A)(in preparation)

Edited by Carl Wu and C Davis Allis

Volume 376 Chromatin and Chromatin Remodeling Enzymes (Part B)(in preparation)

Volume 377 Chromatin and Chromatin Remodeling Enzymes (Part C)(in preparation)

Volume 378 Quinones and Quinone Enzymes (Part A) (in preparation)Edited by Helmut Sies and Lester Packer

Volume 379 Energetics of Biological Macromolecules (Part D)

Edited by Chandan K Sen and Gregg L Semenza

Volume 382 Quinones and Quinone Enzymes (Part B) (in preparation)Edited by Helmut Sies and Lester Packer

Volume 383 Numerical Computer Methods, (Part D) (in preparation)Edited by Ludwig Brand and Michael L Johnson

Volume 384 Numerical Computer Methods, (Part E) (in preparation)Edited by Ludwig Brand and Michael L Johnson

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[1] On Models and Methods for Studying

Polymerase Translocation

By Rui Sousa

When trying to understand how macromolecules move, it is tempting toextrapolate from our everyday experience, and to imagine that they musthave active mechanisms to push or pull themselves along However, it isimportant to keep in mind that at the microscopic level, and at tempera-tures at which water is a liquid, molecular movement is rapid and unceas-ing.1What is required are mechanisms to give direction to this random,Brownian motion Our studies of RNA polymerase translocation havetherefore focused on two questions: (1) How can the random collisionsand jostling that characterize the microscopic world be harnessed to drive

an RNA polymerase in a directed fashion along a template? (2) What arethe consequences of such mechanisms of movement for the enzymology oftranscription and for regulation of polymerase activity?

Model for Polymerase Translocation

In developing a model for RNA polymerase translocation we sought toobey the dictum that mechanism should be as simple as possible (but nosimpler) We imagined a mechanism in which the polymerase would bebound to the DNA in a manner that allows it to be pushed back and forth

by intermolecular collisions.2 At the same time, the requirement forprocessivity in synthesis meant that the polymerase might be able to slide

on the template, but that once engaged in transcript elongation, it shouldn’tdisengage from the DNA until it encounters a terminator Structural stud-ies of polymerases provide a basis for believing in such a binding modesince they reveal that these enzyme have deep template binding cleftssurrounded by flexible elements which might wholly or partially wraparound the DNA so as to allow polymerase sliding while preventingpolymerase dissociation.3–6 Most dramatic in this respect are the DNA

1 A Einstein, Annalen Der Physik 19, 371–381 (1906).

2 R Guajardo and R Sousa, J Mol Biol 265, 8–19 (1997).

3 R Sousa, J Rose, and B C Wang, J Mol Biol 244, 6–12 (1994).

4 D Jeruzalmi and T A Steitz, EMBO J 17, 4101–4113 (1998).

5 G Zhang, E A Campbell, L Minakhin, C Richter, K Severinov, S A Darst, Cell 98, 811–824 (1999).

6 P Cramer, D A Bushnell, R D Kornberg, Science 292, 1863–1876 (2001).

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the central hole.7In addition to structures which keep the polymerase fromreleasing the DNA, we also considered that interactions with DNAand RNA might limit the range of sliding For example, base pairing be-tween DNA and RNA and interactions between RNA and polymerasewould keep the polymerase from sliding away from the 30-end of theRNA in the downstream direction, while extensive upstream sliding could

be restricted if the template binding cleft immediately downstream ofthe active site (which normally binds single-stranded template) is unable

to accommodate an RNA:DNA hybrid We therefore end up with themodel shown in Fig 1A, where the polymerase can slide on the templateover a range that restricts it to primarily occupy two positions: the pre-translocated position, in which the 30-nt of the RNA occupies the site,which is bound by the substrate NTP during catalysis, and the post-translo-cated position, in which the RNA has cleared the NTP binding site This

7 J Kuriyan and M O’Donnell, J Mol Biol 234, 915–925 (1993).

Fig 1 (A) A model for polymerase translocation.9The RNA polymerase is assumed to be able to slide passively back and forth on the DNA template, driven by intermolecular collisions and fluctuations The pre-translocated position corresponds to the position of the polymerase immediately after completion of bond formation: the 3 0 -nucleotide of the RNA occupies the NTP binding site (indicated by the triangle) Forward sliding clears the RNA 3 0 -nt out of the NTP binding site (post-translocated position), allowing NTP binding to occur Because the NTP can only bind to the post-translocated polymerase, the apparent K NTP is equal to: (kd/kb) (1þk r /kf), where kdand kbare, respectively, the rates of NTP dissociation and NTP binding and kfis the rate of sliding from pre- to post-translocated position, while kris the rate of sliding from post- to pre-translocated position Sliding upstream of the pre-translocated position, or downstream of the post-translocated position, can occur and may lead to transcription arrest or disruption of the elongation complex but is disfavored because it would cause loss of RNA:RNAP interaction or RNA:template base-pairing, or lead to unfavorable interactions between RNAP and the RNA:DNA hybrid 2 (B) A DNA bound protein can impede polymerase translocation leading, in turn, to an increase in apparent KNTP.

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model has been tested both by developing and evaluating its kineticimplications, and by monitoring the position of the polymerase duringtranscript elongation with exonucleases.

Kinetic Consequences and Tests of the Model

The NTP can only bind to the transcription complex in the translocated position, i.e., after the RNA has cleared the NTP binding site(Fig 1A) If it is assumed further that movement between pre- and post-translocated positions is faster than the rate of bond formation (a reason-able assumption for macromolecular Brownian motions over distances of1–10 A˚ ), then the effect on the kinetics of the reaction is to increase the ap-parent KNTPby the factor: (1þkr/kf), where kfis the rate of movement frompre- to post-translocated positions, and kris the rate of the reverse move-ment (Fig 1A).2The (1þkr/kf) term should seem familiar: it is analogous tothe (1þ[I](kb/kd)) term that increases the apparent Km of an enzyme inclassic competitive inhibition, where [I] is the inhibitor concentration, kb

post-is the rate of inhibitor binding to enzyme and kd is the rate of inhibitor:enzyme dissociation In the mechanism shown inFig 1Athe competitiveinhibitor may be considered to be the 30-nt of the RNA (which is at a con-centration of unity), while krand kfare analogous to kband kd, the rates ofthe steps that either fill or clear the substrate binding site of inhibitor.The kinetic consequence of this mechanism is that anything which im-pedes the forward movement of the transcription complex (slows kf) willincrease the local apparent KNTP of the NTP which is incorporatedfollowing translocation The simplest and probably most physiologicallyrelevant test of this is to measure KNTP on collision of the transcriptioncomplex with a DNA bound protein (Fig 1B), since such encounters mustoccur frequently in vivo We did this by using a T7 promoter containing lacrepressor binding sites centered at either þ13 or þ15.8

Lac repressorblocked transcript extension beyondþ4 or þ6 when bound to the þ13 orþ15 centered sites, respectively However, transcription extension fromþ3 to þ4, or from þ5 to þ6, was impeded, but not blocked, by repressorbound at the þ13 or þ15 centered sites, respectively.9

This impedancewas measured as an increase in the % abortion (termination) of transcrip-tion occurring at RNA lengths of 3 and 5 nt, relative to what was seen withthe same templates in the absence of bound repressor (Fig 2) Consistentwith the kinetic consequences of the translocation mechanism outlined in

Fig 1, this impedance could be overcome by increasing the concentration

8 P J Lopez, J Guillerez, R Sousa, and M Dreyfus, J Mol Biol 269, 49–51 (1997).

9 R Guajardo, P J Lopez, M Dreyfus, and R Sousa, J Mol Biol 281, 777–792 (1998).

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of the NTP utilized in the extension step (i.e., the NTP used to extend theRNA from 3 to 4 nt, or from 5 to 6 nt) Similar effects were observed when

a barrier to translocation was formed by using NTP limitation to halt aT7RNAP elongation complex atþ13.9

In these experiments barriers were arranged to impede transcriptextension while the polymerase was engaged in ‘‘abortive’’ transcription,

Fig 2 Increased NTP concentrations overcome the obstacle to T7 RNAP translocation created by bound lac repressor.9Lanes 1-8: Transcript patterns from a linearized template containing a T7 promoter The sequences of the transcripts generated during abortive transcription (3–8mers) are given GTP, CTP, UTP are at 5 mM (transcript were labeled by including 32P-GTP in the reaction) ATP concentrations were varied as indicated on the top

of each gel lane Lanes 9-16: As in lanes 1-8 except that lac repressor is present at 0.3 M (30-fold excess over template) The template contains a lac repressor binding site centered at þ15 Bound repressor impedes transcript extension from 5 to 6 nt, as evidenced by an increase

in the amount of 5mer product at low ATP concentrations However, increases in ATP concentrations overcome the impedance due to lac repressor, as evidenced by the decrease in 5mer at high ATP concentrations.

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during which short RNAs are released from the transcription complex as asignificant rate This was done so as to allow impedance to be detected as

an increase in the amount of termination at a particular transcript length(seeFig 2) With appropriately constructed templates such methods could

be used to measure impedance to translocation as a function of [NTP] forany RNA polymerase and DNA binding protein barrier (the situation inwhich pol II encounters a histone is of obvious interest), so long as the bar-rier interferes with translocation during the abortive phase of transcription.Without a quench- or stop-flow apparatus capable of millisecond time reso-lution, it may be difficult to measure apparent KNTP effects during tran-script elongation Consider that transcript extension by E coli or T7RNAP occurs at rates of 10-100 nt/sec or 200 nt/sec, respectively, andthat transcription is highly processive during elongation.10,11 In a manualexperiment, it would not be possible to detect impedance that, for example,slows a single nucleotide extension step by a factor of 10 However, thefeasibility of characterizing E coli RNAP kinetics with millisecond timeresolution using quench-flow methods has been recently demonstrated.12Using such methods, together with appropriate templates that would place

a DNA bound protein or other obstacle in front of an elongation complex,

it should be possible to determine whether an obstacle that impedes—butdoes not completely block—translocation causes the type of local increases

in apparent KNTPpredicted from the mechanism inFig 1

The kinetic consequences of this mechanism are also relevant to molecule studies in which RNAPs transcribe against an applied force Suchstudies have generally found that tugging on the RNAP does not slow itdown, unless one tugs hard enough to disrupt and arrest the complex.13,14This is consistent with the translocation mechanism shown in Fig 1, be-cause in this mechanism, translocation is a rapid, diffusive step (i.e., it isnot rate-limiting for extension), so slowing translocation down (by tugging

single-on the polymerase) does not slow the overall rate of extensisingle-on However,the mechanism inFig 1predicts that the effects of such tugging will be felt

as increase in apparent KNTP Therefore, single-molecule studies of scription against an applied force should look not at whether transcription

tran-is slowed at high NTP concentrations, but at whether the application of

10 O V Makarova, E M Makarov, R Sousa, M Dreyfus, Proc Natl Acad Sci USA 92, 12250–12254 (1995).

11 J Huang, J Villemain, R Padilla, and R Sousa, J Mol Biol 293, 457–475 (1999).

12 J E Foster, et al., Cell 106, 242–252 (2001).

13 M D Wang, M J Schnitzer, H Yin, R Landick, J Gelles, and S M Block, Science 282, 902–906 (1998).

14 A D Mehta, M Rief, J A Spudich, D A Smith, and R M Simmons, Science 283,

1689 (1999).

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normal KNTPof the enzyme.

Using Exonucleases to Study Translocation

Kinetic effects rarely allow for unambiguous interpretation of ism, so it is important to use a more direct probe of polymerase position totest models for translocation mechanism Exonucleases may be more usefulthan area footprinting reagents in this respect because a single nucleotideshift in the position of a diffuse footprint that extends over 20 or 30 nucle-otides can be difficult to observe, while a similar shift in the position atwhich digestion by an exonuclease is blocked is readily detected (Fig 3)

mechan-We used exonuclease III (which digests in the 30 to 50 direction) todetect the upstream and downstream boundaries of T7RNAP elongationcomplexes on 50-end labeled template or nontemplate DNA strands,respectively Lambda exonuclease (which digests in the 50to 30 direction)

Fig 3 Exonuclease III mapping of the template strand upstream boundary of T7 RNAP elongation complexes as a function transcript length and NTP binding15: Elongation complexes with 3 0 -dNMP terminated transcripts were formed on templates in which the

5 0 -end of the template strand was 32P-labeled NTPs were removed by ultrafiltration Transcripts ranged from 14 to 17 nts in length, as indicated over each gel lane NTPs complementary to the template base immediately downstream of the RNA 30-end were added back to the complexes as indicated in lanes 4, 6, and 8 Complexes were treated with 1 u/l of Exo III for 15 min at r.t The template strand sequence from þ1 to þ18 is given on the left The bands between 1 and þ6 in lanes 2-8 show where exo III digestion halts due to collision with the upstream boundary of the elongation complex Lane 1 contains a marker derived from restriction digestion DNA lengths are given on the right.

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was used to detect upstream and downstream boundaries on 30-end labelednon-template or template DNA strands, respectively.15 To evaluate theeffects of NTP binding on translocation we prepared elongation complexes

in which the transcript was 30-terminated by incorporation of a 30-dNMP(30-dNTPs are available from Trilink Biotechnologies (T7RNAP readily in-corporates 30-dNMPs, but it is not known how readily E coli RNAP or pol

II will incorporate these chain terminators) Excess NTPs were thenwashed away by ultrafiltration, using Amicon microcon ultrafiltration unitswith 100 kD cutoff membranes (other methods such as using His-taggedRNAPs or biotinylated DNAs immobilized on solid supports would prob-ably work as well) The position of the elongation complex, as monitored

by exonuclease digestion, was then examined in the absence or presence

of saturating concentrations of the NTP complementary to the templatebase immediately downstream of the RNA 30-end (addition of a non-complementary NTP provides a control for non-specific effects) As seen

in Fig 3, downstream movement of the elongation complex is seen, notupon completion of the transcript extension step, but upon NTP binding(i.e., the complex with the 15mer þ UTP in lane 4 is at nearly the sameposition as the complex with 16mer in lane 5) This is consistent with thetranslocation mechanism inFig 1, and with the additional conclusion that,

in the rapid equilibrium between pre- and post-translocated positions, thepre-translocated position is favored until the NTP binds to and stabilizesthe post-translocated complex.2,15

Because the elongation complexes can slide they do not present animpenetrable barrier to the processive action of exonucleases It is there-fore essential that a range of exonuclease concentrations and/or digestiontimes be evaluated so as to identify appropriate digestion conditions It isalso critical that comparisons only be made between elongation complexes

in parallel reactions with identical exonuclease concentrations and tion times Exonuclease III is better for mapping the actual boundaries ofthe elongation complex since it is able to digest almost to the edge of thecomplex, while lambda exonuclease stops 8 nt away from the complex.15This probably reflects the position of the exonuclease active site relative

diges-to its leading edge In addition diges-to effects on the position of the complex,NTP binding increases the resistance of the elongation complex to sliding.This is especially apparent at the downstream boundary of the elongationcomplex, where extended digestion with lambda exonuclease can push theboundary of the complex lacking NTP upstream (Fig 4) However, whenthe NTP binds to the complex it becomes much more resistant to theprocessive action of the exonuclease (seeFig 4)

15 J Huang and R Sousa, J Mol Biol 303, 347–358 (2000).

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The resistance to sliding seen upon binding NTP may be directly due totemplate:NTP:RNAP interactions, or to an NTP-induced isomerizationthat causes the polymerase to close in around the template Johnson out-lined a translocation mechanism for DNAPs in which the polymerase, inthe absence of bound NTP, assumes an ‘‘open’’ conformation, which canslide passively on the DNA.16 NTP binding induces isomerization to a

‘‘closed’’ conformation, which is resistant to sliding These NTP bindingdriven transition—initially inferred from kinetic studies—were subse-quently observed directly in crystal structures of DNAPs with and withoutbound NTPs.17,18T7 RNAP is highly homologous to the DNAP I family19and probably follows a similar mechanism

Exonuclease digestion and other footprinting reagents have also beenused to obtain evidence for sliding by E coli RNAP and pol II.20–22Though

16 K A Johnson, Annu Rev Biochem 62, 685–713 (1993).

17 S Doublie´, S Tabor, A M Long, C C Richardson, and T Ellenberger, Nature 391, 251–258 (1998).

18 Y Li, S Korolev, and G Waksman, EMBO J 17, 7514–7525 (1998).

19 R Sousa, Trends in Biochem Sci 21, 186–190 (1996).

20 R Landick, Cell 88, 741–744 (1997).

21 N Komissarova and M Kashlev, J Biol Chem 272, 15329–15338 (1997).

22 N Komissarova and M Kashlev, Proc Natl Acad Sci USA 25, 14699–14704 (1998).

Fig 4 Lambda exonuclease mapping of the template strand downstream boundary of a T7 RNAP elongation complex with a 3 0 -dNMP terminated 15 nt RNA 15 A: The NTP complementary to the template base immediately downstream of the RNA 3 0 -end (UTP) was added to the reactions at concentrations indicated over each gel lane Complexes were digested with either 0.4 (lanes 1-12) or 0.08 (lanes 13-24) u/l of lambda exonuclease for

60 min at r.t The downstream boundary of the ternary complex (DNAþRNAPþRNA) is diffuse and is pushed upstream at higher exonuclease concentrations The quaternary complex (DNAþRNAPþRNAþNTP) is more resistant to the processive exonuclease, as evidenced by

a sharper boundary, which is not pushed back at the higher exonuclease concentration B: Control for nonspecific NTP effects Digestion done as in lanes 13-24 detects only the ternary complex boundary if non-complementary NTP (GTP) is added.

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strict quantitative comparisons have not been done, the multisubunitRNAPs seem more prone than T7 RNAP to engage in long-range upstreamsliding in which the transcription bubble and RNA:DNA hybrid track theposition of the polymerase and the 30-end of the RNA is displaced fromthe active site.20Such sliding can result in formation of arrested complexesthat require RNA cleavage to resume elongation.21,22While these observa-tions are supportive of the general idea that RNAPs can slide on their tem-plates, it must be kept in mind that these longer-range sliding events thatlead to formation paused or arrested elongation complexes are unlikely to

be in rapid equilibrium with the pre- and post-translocated states illustrated

inFig 1and therefore, they will not increase local KNTP,app Instead, escapefrom the paused or arrested state becomes locally rate-limiting.23

Studying Translocation with RNAses

Translocation of the polymerase can also be monitored with RNAse,because downstream movement of the polymerase relative to the RNAexposes more RNA to digestion Experiments are done as described forthe exonuclease digestions: ECs are prepared with 30-dNMP terminatedRNAs, NTPs are washed away, and then digestion is monitored as a func-tion of the presence of the NTP complementary to the template base im-mediately downstream of the RNA 30-end (Fig 5).15 The RNA can belabeled at either the 50

30-end by using an appropriately designed template (we used templateswhere C and U were incorporated only atþ15 or þ16,24so that transcriptsterminated at þ16 or þ17 could be 30-end labeled by inclusion of -32PCTP or UTP in the transcription reaction) As with the exonuclease diges-tions, different RNAse concentrations and digestion times must be tested,and any comparisons must be made between ECs treated in parallel underidentical conditions Translocation induced by NTP binding is detected as

an increase in the RNAse sensitivity of the RNA at the point where itemerges from the transcription complex (seeFig 5)

Future Studies

The methods described here, which have been used to study T7 RNAPtranslocation, can be readily adapted to study translocation by otherpolymerases An important question will be whether cells take advantage

23 P Urali and R Landick, J Mol Biol 311, 265–282 (2001).

24 P E Mentesanas, S T Chin-Bow, R Sousa, and W T McAllister, J Mol Biol 302, 1049–1062 (2000).

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of the kinetics of these processes to create mechanisms for regulation It issometimes said that steps in a reaction which are not rate-limiting are ‘‘kin-etically invisible.’’ This seems to suggest that they are irrelevant for control

or regulation Nothing could be less true Polymerase translocation doesnot appear to be rate-limiting during either RNA2,13,14or DNA16synthesis,but as emphasized here it can affect the reaction by its effect on apparent

KNTP(for that matter, the substrate binding and dissociation steps in anyrapid equilibrium enzyme reaction are ‘‘kinetically invisible’’ because they

Fig 5 A: RNAse T1 sensitivity of a T7 RNAP elongation complex with a 3 0 -dNMP terminated 15 nt RNA 15 Transcript sequence is complementary to the þ1 to þ15 template strand sequence given in Fig 3 The transcript is labeled by inclusion of 32 P-GTP in the reaction Transcript lengths are given on the right of the gel Elongation complexes were treated with 0 (lane 1), 0.5 (lanes 2, 7), 0.25 (lanes 3, 8), 0.15 (lanes 4, 9), 0.075 (lanes 5, 10), or 0.03 (lanes 6, 11) u/l RNAse T1 for 10 min at r.t with either 0 (lanes 1-6) or 0.5 mM (lanes 7-11) of the complementary NTP (UTP) present B: Scans of lanes 6 (left) and 11 (right) of the gel shown in A Increased digestion 12 nt away from the RNA 3 0 -end in the presence of UTP indicates forward translocation of the polymerase and consequent increased exposure of RNA

12 nt away from the 3 0 -end upon NTP binding to the elongation complex C: Interpretation of the exonuclease and RNAse digestions patterns The polymerase slides between pre- and post-translocated positions—spending most of its time in the pre-translocated position— unless NTP binds to the post-translocated complex In the post-translocated complex, DNA (which is protected in the pre-translocated complex) at the back end (‘‘B’’) becomes acessible, while DNA at the front end (‘‘F’’) becomes covered In addition, RNA 12 nt away from the

3 0 -end (‘‘12’’) is protected in the pre-translocated complex but acessible in the translocated complex NTP binding therefore leads to a downstream shift in exo III ( Fig 3 ) and lambda exo ( Fig 4 ) digestion, and increased RNAse accessibility of the RNA 12 nt from the 3 0 -end ( Fig 5 ).

Tiêu đề	RNA Polymerase and Associated Factors, Part D
Trường học	California Institute of Technology
Chuyên ngành	Biology
Thể loại	Article
Thành phố	Pasadena

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