rna polymerase and associated factors, part c

Nucleic Acids and Protein Synthesis Part C Edited by Kivie Moldave and Lawrence Grossman Volume XXI.. Nucleic Acids and Protein Synthesis Part E Edited by Lawrence Grossman and Kivie Mol

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It has been just seven years since Volumes 273 and 274 of Methods in mology, which covered RNA polymerase and associated activities, were pub-lished Since then, there has been an explosion in the amount of informationthat has come out on RNA polymerase and transcription, driven by theintensification in technology In Volumes 370 and 371, we try to take up whereVolumes 273 and 274 left off, but we include and emphasize what we feel is animportant aspect of RNA polymerase that was only touched on in the previousvolumes It is quite clear now that RNA polymerase does not act alone Itsassociated factors are key in determining initiation, elongation, and termin-ation occurrences The reliance on the associated factors is often so great that it

Enzy-is difficult to determine whether a given protein Enzy-is an RNA associated factor or is, in fact, a subunit of the enzyme

polymerase-The chapters in these volumes describe the RNA polymerase enzymes andthe associated factors, and their effects on transcription initiation, elongation,and termination The chapters expose both prokaryotic and eukaryotic systems,but they are purposely kept undivided irrespective of the origins of the RNApolymerases We predict that as more is learned about the prokaryotic andeukaryotic systems of transcription and gene regulation, we will realize howsimilarly they behave rather than how differently

Sankar AdhyaSusan Garges

xix

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METHODS IN ENZYMOLOGY

EDITORS-IN-CHIEF

DIVISION OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY

PASADENA, CALIFORNIA

FOUNDING EDITORSSidney P Colowick and Nathan O Kaplan

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Article numbers are in parentheses and following the names of contributors.

Affiliations listed are current.

Annie-Claude Albert (11), Biology

Department, Washington University in

St Louis, One Brookings Drive, St.

Louis, Missouri 63130

J.-F Allemand (49), Laboratoire de

Phys-ique StatistPhys-ique, CNRS UMR 8550,

Ecole Normale Superiure, 24 rue

Lho-mond, 75231 Paris, France

Jennifer R Anthony (5), Department of

Bacteriology, University of Wisconsin –

Madison, 1550 Linden Drive, Madison,

Wisconsin 53706

Larry C Anthony (16), McArdle

Labora-tory of Cancer Research, University of

Wisconsin, 1400 University Avenue,

Madison, Wisconsin 53706

Jae-Bum Bae (7), Laboratory of Molecular

Microbiology, School of Biological

Sci-ences, Seoul National University, Seoul

151-742, Korea

Vladimir B Bajic (21), Laboratories for

Information Technology, 21, Heng Mui

Keng Terrace, Singapore 119613

Rajiv P Bandwar (55), Department

Biochemistry, UMDNJ, Robert Wood

Johnson Medical School, 675 Hoes Lane,

Piscataway, New Jersey 08854

Peter B Becker (42),

Adolf-Butenandt-Institut, Molekularbiologie,

Ludwig-Maximilians-Universitat, Schillerstrasse

44, D80336 Munchen, Germany

Veit Bergendahl (17), McArdle

Labora-tory of Cancer Research, University of

Sukesh R Bhaumik (38), Department of Biochemistry and Molecular Biology, Southern Illinois University, Carbondale, Illinois 62901

Vladimir Bondarenko (29), Department

of Biochemistry and Molecular Biology, Wayne State University School of Medi- cine, Detroit, Michigan 48201

Patricia Bordes (53), Department of Biological Sciences, National College of Science, Technology, and Medicine, SAFB, Imperial College Road, London SW7 2AZ, United Kingdom

Robert Britton (23), Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824

Vladimir Brusic (21), Laboratories for Information Technology, 21, Heng Mui Keng Terrace, Singapore 119613 Martin Buck (3, 53), Department of Bio- logical Sciences, Wolfson Laboratories, Imperial College of London, Rm 313, London SW7 2AY, United Kingdom Richard Burgess (16, 17), McArdle Laboratory of Cancer Research, Univer- sity of Wisconsin, 1400 University Avenue, Madison, Wisconsin 53706 Patricia C Burrows (3), Department of Biological Sciences, Wolfson Laborator- ies, Imperial College of London, Rm 313, London SW7 2AY, United Kingdom Julio Cabrera (1), Laboratory of Molecu- lar Biology, National Cancer Institute, National Institutes of Health, Bldg 37,

Rm 5144, Bethesda, Maryland 20892

xi

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Wendy Cannon (53), Department of

Biological Sciences, National College of

Science, Technology, and Medicine,

SAFB, Imperial College Road, London

SW7 2AZ, United Kingdom

Michael W Capp (45), Department of

Chemistry, University of Wisconsin, 433

Babcock Drive, Madison, Wisconsin

53706-1544

Frederic Coin (58), Institut de Genetique

et de Biologie Moleculaire et Cellulaire,

Dept of Transcription, CNRS/INSERM/

ULP, B.P 162, 67404 Illkirch Cedex,

C.U de Strasbourg, France

Ronald C Conaway (59), Dept of

Bio-chemistry and Molecular Biology, Kansas

University Medical Center, Kansas City,

Missouri 66160

Benoit Coulombe (57), Laboratory of

Gene Transcription, Clinical Research

In-stitute of Montreal, 110 Pine Avenue

West, Montreal, Quebec H2W 1R7,

Canada

V Croquette (49), Laboratoire de

Phys-ique StatistPhys-ique, CNRS UMR 8550,

Ecole Normale Superiure, 24 rue

Lho-mond, 75231 Paris, France

Michael E Dahmus (13), Section of

Mo-lecular and Cell Biology, Division of

Bio-logical Science, University of California,

Davis, Davis, California 95616-5224

Seth A Darst (4), The Rockefeller

Univer-sity, 1230 York Avenue, Box 224, New

York, New York 10021

Dipak Dasgupta (50), Biophysics Division,

Saha Institute of Nuclear Physics, 37,

Belgachia Road, Calcutta 700037, India

Margaret A Daugherty (31),

Depart-ment of Biochemistry, University of

Ver-mont College of Medicine, Burlington,

Vermont 05405

Elena K Davydova (8), Department of Molecular Genetics and Cell Biology, University of Chicago, 920 E 58th Street, Chicago, Illinois 60637

Pieter deHaseth (47), Department of chemistry, Case Western Reserve University, Cleveland, Ohio 44106-4935 Borries Demeler (43), Center for Analyt- ical Ultracentrifugation of Macromolecu- lar Assemblies, University of Texas Health Science Center, San Antonio, Texas Jonathan A Dodd (10), Department of Biological Chemistry, University of Cali- fornia – Irvine, 240 D Medical Sciences I, Irvine, California 92697-1700

Bio-Timothy J Donohue (5), Department of Bacteriology, University of Wisconsin – Madison, 1550 Linden Drive, Madison, Wisconsin 53706

David Dunlap (32), DIBIT 3A3, san faele Scientific Institute, via Olgettina,

Raf-58, Milan 20132, Italy Arik Dvir (59), Department of Biological Sciences, Oakland University, Rochester, Michigan 48309

Keith Earley (11), Biology Department, Washington University in St Louis, One Brookings Drive, St Louis, Missouri 63130

R H Ebright (49), Howard Hughes ical Institute, Waksman Institute, De- partment of Chemistry, Rutgers University, Piscataway, New York 08854 Jean Marc Egly (58), Institut de Genetique

Med-et de Biologie Moleculaire Med-et Cellulaire, Department of Transcription, CNRS/IN- SERM/ULP, B.P 162, 67404 Illkirch Cedex, C.U de Strasbourg, France Robert D Finn (3), The Wellcome Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom

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Laura Finzi (32), Dip Biol ‘‘Luigi Gorini,’’

U Degli Studi Milano, Via Celoria 26,

Milan I-20133 Italy

Katherine M Foley (16), McArdle

Lab-oratory of Cancer Research, University of

Diane Forget (57), Laboratory of Gene

Transcription, Clinical Research Institute

of Montreal, 110 Pine Avenue West,

Montreal, Quebec H2W 1R7, Canada

Michael G Fried (31), Department of

Biochemistry and Molecular Biology,

The Pennsylvania State University

Col-lege of Medicine, 500 University Drive,

Hershey, Pennsylvania 17033

Susan Garges (25), Laboratory of

Mo-lecular Biology, CCR, National Cancer

Institute, National Institutes of Health,

Bldg 37, Rm 5138, Bethesda, Maryland

20892

S Gilfillan (40), GSF-National Research

Center for Environment and Health,

De-partment of Gene Expression, Institute of

Molecular Immunology, Marchionini-str.

25, Munich D-81377, Germany

James A Goodrich (56), Department of

Chemistry and Biochemistry, University

of Colorado at Boulder, Boulder,

Color-ado 80309-0215

Richard L Gourse (51), Department of

Wisconsin 53706-1567

Brent Gowen (3), 627 Gower Point Road,

Gibson’s Landing, British Columbia V0N

1V8, Canada

Heather A Green (5), Department of

Wisconsin 53706

Michael R Green (36, 38), Program in Gene Function and Expression, 364 Plan- tation Street, Worcester, Massachusetts 01605-2377

Jack Greenblatt (34), Banting and Best Department of Medical Research, Univer- sity of Toronto, Toronto, Ontario M5G 1L6, Canada

Carol A Gross (18), Departments of matology and Microbiology and Immu- nalogy, University of California, San Francisco, California 94143-0512

Sto-Tanja M Gruber (18), Departments of Stomatology and Microbiology and Im- munalogy, University of California, San Francisco, California 94143-0512

Mi-Young Hahn (7), Laboratory of Molecular Microbiology, School of Biological Sciences, Seoul National Uni- versity, Seoul 151-742, Korea

Mitsuhiro Hamada (14), Department of Integrated Biosciences, University of Tokyo, Tokyo, Japan

John D Helmann (2), Department of Microbiology, Cornell University, Ithaca, New York 14853

Susanne Hoheisel (34), Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, 401 Barker Hall, Berkeley, Californaia 94720-3202

Ying Huang (14), Laboratory of lar Growth Regulation, National Institute

Molecu-of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-2753

Diane Imburgio (19), Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, 190 Frelinghuysen Road, Piscataway, New Jersey 08854

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Ding Jun Jin (1, 15, 25, 26), Laboratory of

Molecular Biology, National Cancer

In-stitute, National Institutes of Health,

Bldg 37, Rm 5144, Bethesda, Maryland

20892

Susan Jones (53), Department of

Bio-logical Sciences, National College of

Sci-ence, Technology, and Medicine, SAFB,

Imperial College Road, London SW7

2AZ, United Kingdom

Caroline M Kane (34), Department of

Molecular and Cell Biology, Division of

Biochemistry and Molecular Biology, 401

Barker Hall, Berkeley, California

94720-3202

Changwon Kang (54), Department of

Bio-logical Sciences, Korea Advanced

Institute of Science and Technology,

373-1 Guseong-dong, Yuseong-gu, Daejon

305-701, Republic of Korea

Mikhail Kashlev (12), NCI Center for

Cancer Research, National Cancer

Insti-tute, Frederick Cancer Research and

Development Center, Bldg 539, Rm.

222, Frederick, Maryland 21702

Krystyna M Kazmierczak (8),

Depart-ment of Molecular Genetics and Cell

Biol-ogy, University of Chicago, 920 E 58th

Street, Chicago, Illinois 60637

Patricia J Kiley (27), Department of

Bio-molecular Chemistry, University of

Wis-consin, 574 Medical Science Center, 1300

University Avenue, Madison, Wisconsin

53706

Maria L Kireeva (12), NCI Center for

Cancer Research, National Cancer

Insti-tute, Frederick Cancer Research and

De-velopment Center, Bldg 539, Rm 222,

Frederick, Maryland 21702

Bruno P Klaholz (3), Institut de

Genet-ique et de Biologie Moleulaire et

Celle-laire, Dept de Biologie et Genomique

Structurales, 1, rue Laurent Fries, 67404

Illkirch, France

Michael S Kobor (34), Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, 401 Barker Hall, Berkeley, California 94720- 3202

Natalia Komissarova (12), NCI Center for Cancer Research, National Cancer Institute, Frederick Cancer Research and Development Center, Bldg 539, Rm 222, Frederick, Maryland 21702

E Kremmer (40), GSF-National Research Center for Environment and Health, De- partment of Gene Expression, Institute of Molecular Immunology, Marchionini-str.

25, Munich D-81377, Germany Jennifer F Kugel (56), Department of Chemistry and Biochemistry, University

of Colorado at Boulder, Boulder, ado 80319-0215

Color-Konstantin Kuznedelov (9), ogical Institute of the Russian Academy

Limnol-of Sciences, Irkutsk, Russia

Lester J Lambert (43), Laboratory of Molecular Biophysics, The Rockefeller University, New York, New York 10021

Dale E A Lewis (52), Laboratory of lecular Biology, National Cancer Insti- tute, 37 Convent Drive, Rm 5138, Bethesda, Maryland 20892-4255

Mo-Patrick S Lin (13), Section of Molecular and Cell Biology, Division of Biological Science, University of California, Davis, Davis, California 95616-5224

Ye V Liu (29), Department of try and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201

Biochemis-Lucyna Lubkowska (12), NCI Center for Cancer Research, National Cancer Insti- tute, Frederick Cancer Research and De- velopment Center, Bldg 539, Rm 222, Frederick, Maryland 21702

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Betina Macho (35), Institut de Genetique

et de Biologie Moleculaire et Cellulaire,

1, rue Laurent Fries, 67404 Illkirch,

Strasbourg, France

Richard Maraia (14), Laboratory of

Mo-lecular Growth Regulation, National

Institute of Child Health and Human

Development, National Institutes of

Health, Bethesda, Maryland 20892-2753

Robert G Martin (24), Laboratory of

Molecular Biology, NIDDK, National

Institutes of Health, Bethesda, Maryland

20892-0560

Shoko Masuda (4), The Rockefeller

Uni-versity, 1230 York Avenue, Box 224, New

York, New York 10021

Kalai Mathee (22), Department of

Bio-logical Sciences, Florida International

University, Miami, Florida 33199

Walter Messer (30),

Max-Planck-Insti-tute for Molecular Genitics, D-14195

Berlin, Germany

M Miesterernst (40), GSF-National

Re-search Center for Environment and

Health, Department of Gene Expression,

Institute of Molecular Immunology,

Marchionini-str 25, Munich D-81377,

Germany

Leonid Minakhin (9), Waksman Institute

of Microbiology, Rutgers, The State

Uni-versity of New Jersey, 190 Frelinghuysen

Road, Piscataway, New Jersey 08854

Aaron P Mitchell (41), Department of

Microbiology and Institute of Cancer

Re-search, Columbia University, 701 West

168th Street, New York, New York 10032

Katsuhiko S Murakami (4), The

Rocke-feller University, 1230 York Avenue, Box

224, New York, New York 10021

Heath D Murray (51), Department of

Wisconsin 53706-1567

Yoshihiro Nakatani (37), Dana-Farber Cancer Institute and Harvard Medical School, Boston, Maryland 02115 Giri Narasimhan (22), School of Com- puter Science, ECS 389, Florida Inter- national University, Miami, Florida 33199

Sergei Nechaev (19, 53), CMG, University

of California San Diego, 9500 Gilman Drive, La Jolla, California 92093 Yur A Nedialkov (44), Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319

Alexander J Ninfa (29), Department of Biochemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606

Masayasu Nomura (10), Department of Biological Chemistry, University of Cali- fornia – Irvine, 240 D Medical Sciences I, Irvine, California 92697-1700

Michael C O’Neill (20), Department of Biological Sciences, University of Mary- land, Baltimore County (UMBC), 1000 Hilltop Circle, Baltimore, Maryland 21250

Vasily Ogryzko (37), Laboratoire genese, Differenciation et Transduction

Onco-du Signal, CNRS UPR 9079, Institut Andre Lwoff, Villejuif, France

Elena V Orlova (3), Department of tallography, Birbeck College, Malet Street, London WC1E 7HX, United Kingdom

Crys-Joo-Hong Park (7), Laboratory of Molecular Microbiology, School of Biological Sciences, Seoul National Uni- versity, Seoul 151-742, Korea

Smita S Patel (55), Department istry, UMDNJ, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscat- away, New Jersey 08854

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Biochem-Erik Pierstorff (34), Department of

Molecular and Cell Biology, Division of

Biochemistry and Molecular Biology, 401

Barker Hall, Berkeley, California

94720-3202

Craig S Pikaard (11), Biology

Depart-ment, Washington University in St Louis,

One Brookings Drive, St Louis, Missouri

63130

Aparna Raval (33), Experimental

Immunology Branch, National Cancer

Bldg 10, Rm 4B36, Bethesda, Maryland

20892-1360

Pampa Ray (3), Department of Biological

Sciences, Wolfson Laboratories, Imperial

College of London, Rm 313, London

SW7 2AY, United Kingdom

M Thomas Record Jr (45), Department

of Chemistry, University of Wisconsin,

433 Babcock Drive, Madison, Wisconsin

53706-1544

Joseph C Reese (36), Deptartment of

Bio-chemistry and Molecular Biology,

Penn-sylvania State University, 203 Althouse

Laboratory, University Park,

Pennsylva-nia 16802

John N Reeve (6), Department of

Microbiology, Ohio State University,

Columbus, Ohio 43210

A Revyakin (49), Howard Hughes

Med-ical Institute, Waksman Institute,

Depart-ment of Chemistry, Rutgers University,

Piscataway, New York 08854

Vikas Rishi (39), Laboratory of

Metabol-ism, National Cancer Institute, National

Institutes of Health, Bldg 37, Rm 2D24,

Bethesda, Maryland 20892

Jung-Hye Roe (7), Laboratory of

Molecu-lar Microbiology, School of Biological

Sciences, Seoul National University,

Seoul 151-742, Korea

Judah L Rosner (24), Laboratory of

Mo-lecular Biology, NIDDK, National

Insti-tutes of Health, Bethesda, Maryland

20892-0560

Lucia B Rothman-Denes (8), ment of Molecular Genetics and Cell Biol- ogy, University of Chicago, 920 E 58th Street, Chicago, Illinois 60637

Depart-Siddhartha Roy (46, 48), Department of Biophysics, Bose Institute, P-1/12, C.I.T., Scheme VII M, Calcutta 700 054, India Ruth M Saecker (45), Department of Chemistry, University of Wisconsin, 433 Babcock Drive, Madison, Wisconsin 53706-1544

Julio Saez-Vasquez (11), Biology ment, Washington University in St Louis, One Brookings Drive, St Louis, Missouri 63130

Raphael Sandaltzopoulos (42), ment of Molecular Biology, Democritus University of Thrace, University Hospital

Depart-at Dragana, G-68100 Alexandroupolis, Greece

Paola Sassone-Corsi (35), Institut de netique et de Biologie Moleculaire et Cel- lulaire, 1, Rue Laurent Fries, 67404 Illkirch, Strasbourg, France

Ge-Virgil Schirf (43), Center for Analytical Ultracentrifugation of Macromolecular Assemblies, University of Texas Health Science Center, San Antonio, Texas David A Schneider (51), Department of Bacteriology, University of Wisconsin – Madison, 1550 Linden Drive, Madison, Wisconsin 53706-1567

Ranjan Sen (50), Laboratory of tion Biology, Center for DNA Finger- printing and Diagnosis, ECIL Road, Nacharam, Hyderabad 500076, India Steve D Seredick (28), Department of Microbiology and Immunology, Univer- sity of British Columbia, 6174 University Boulevard, Vancouver, British Columbia V6T 123, Canada

Transcrip-Konstantin Severinov (9, 19, 53), ment of Molecular Biology and Biochem- istry, Rutgers, The State University of New Jersey, 190 Frelinghuysen Road, Piscataway, New Jersey 08854

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Mitsuhiro Shimizu (41), Deptartment of

Chemistry, Meisei University, Tokyo

191-8506, Japan

Inkyung Shin (54), Department of

Bio-logical Sciences, Korea Advanced

Insti-tute of Science and Technology, 373-1

Guseong-dong, Yuseong-gu, Daejon

305-701, Republic of Korea

Dean D Shooltz (44), Department of

Bio-chemistry and Molecular Biology,

Mich-igan State University, East Lansing,

Michigan 48824-1319

Dinah S Singer (33), Experimental

Immunology Branch, National Cancer

Bldg 10, Rm 4B36, Bethesda, Maryland

20892-1360

George B Spiegelman (28), Department

of Microbiology and Immunology,

Uni-versity of British Columbia, 6174

Univer-sity Boulevard, Vancouver, British

Columbia V6T 123, Canada

G Stelzer (40), GSF-National Research

Center for Environment and Health,

De-partment of Gene Expression, Institute of

Molecular Immunology, Marchionini-str.

25, Munich D-81377, Germany

T R Strick (49), Cold Spring Harbor

Lab-oratory, Cold Spring Harbor, New York

11724

Vasily M Studitsky (29), Department of

Biochemistry and Molecular Biology,

Wayne State University School of

Medi-cine, Detroit, Michigan 48201

Maxim V Sukhodolets (25),

Developmen-tal Genetics Section, Laboratory of

Mo-lecular Biology, CCR, National Cancer

Bldg 37, Rm 5138 Bethesda, Maryland

20892

Victoria Sutton (27), Department of

Bio-molecular Chemistry, University of

Wis-consin, 574 Medical Science Center, 1300

University Avenue, Madison, Wisconsin

Steven J Triezenberg (44), Department

of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319

Oleg V Tsodikov (45), Harvard Medical School, Boston, Massachusetts 02115

Laura Tsujikawa (47), Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106-4935

Barb M Turner (28), Department of Microbiology and Immunology, Univer- sity of British Columbia, 6174 University Boulevard, Vancouver, British Columbia V6T 123, Canada

Marin van Heel (3), Department of Biological Sciences, Wolfson Laborator- ies, Imperial College of London, Rm 313, London SW7 2AY, United Kingdom

Charles Vinson (39), Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Building

37, Rm 2D24, Bethesda, Maryland 20892

Christoph Weigel (30), Institute for Molecular Genitics, D-14195 Berlin, Germany

Max-Planck-Jocelyn D Weissman (33), Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bldg 10, Rm 4B36, Bethesda, Maryland 20892-1360

Joan Weliky Conaway (59), Stowers stitute of Medical Research, 1000 E 50th Street, Kansas City, Missouri 64110

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In-Milton H Werner (43), Laboratory of

Molecular Biophysics, The Rockefeller

University, New York, New York 10021

Siva R Wigneshweraraj (53),

Depart-ment of Biological Sciences, National

College of Science, Technology, and

Medicine, SAFB, Imperial College Road,

London SW7 2AZ, United Kingdom

Yunwei Xie (6), Department of

Microbiol-ogy, Ohio State University, Columbus,

Ohio 43210

Wenxue Yang (26), Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bldg 37, Rm 5144, Bethesda, Maryland 20892

Huijun Zhi (15, 26), Laboratory of lecular Biology, National Cancer Insti- tute, National Institutes of Health, Bldg.

Mo-37, Rm 5144, Bethesda, Maryland 20892

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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

xxi

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Volume XVI Fast Reactions

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 LIV Biomembranes (Part E: Biological Oxidations)

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 90 Carbohydrate Metabolism (Part E)

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 VunakisVolume 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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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, 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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Volume 158 Metalloproteins (Part A)

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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Volume 193 Mass Spectrometry

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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Volume 226 Metallobiochemistry (Part C: Spectroscopic and PhysicalMethods 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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Volume 262 DNA Replication

Edited by Judith L Campbell

Volume 263 Plasma Lipoproteins (Part C: Quantitation)

Edited by William A Bradley, Sandra H Gianturco, and Jere P SegrestVolume 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)Edited by Lester Packer

Volume 270 High Resolution Separation and Analysis of Biological

Macromolecules (Part A: Fundamentals)

Edited by Barry L Karger and William S Hancock

Volume 271 High Resolution Separation and Analysis of Biological

Macromolecules (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 299 Oxidants and Antioxidants (Part A)

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 335 Flavonoids and Other Polyphenols

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)

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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Volume 371 RNA Polymerases and Associated Factors (Part D)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)Edited by Carl Wu and C Davis Allis (in preparation)

Volume 376 Chromatin and Chromatin Remodeling Enzymes (Part B)Edited by Carl Wu and C Davis Allis (in preparation)

Volume 377 Chromatin and Chromatin Remodeling Enzymes (Part C)Edited by Carl Wu and C Davis Allis (in preparation)

Volume 378 Quinones and Quinone Enzymes (Part A)

Edited by Helmut Sies and Lester Packer (in preparation)

Volume 379 Energetics of Biological Macromolecules (Part D)Edited by Jo M Holt, Michael L Johnson, and Gary K AckersVolume 380 Energetics of Biological Macromolecules (Part E)

Edited by Jo M Holt, Michael L Johnson, and Gary K AckersVolume 381 Oxygen Sensing

Edited by Chandan K Sen and Gregg L Semenza

Volume 382 Quinones and Quinone Enzymes (Part B)

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] Construction, Purification, and Characterization of Escherichia coli RNA Polymerases Tagged with Different

Fluorescent Proteins

By Julio E Cabrera and Ding Jun Jin

The green fluorescent protein (GFP) from Aequorea victoria and itscolor variants, the cyan fluorescent protein (CFP) and the yellow fluores-cent protein (YFP), have been widely used in cellular and molecular biol-ogy studies.1–3These proteins are intrinsically fluorescent in a wide range

of hosts and, in most of the cases, remain fluorescent when fused to a targetprotein Some of these fusion proteins have both the physical properties

of the fluorescent proteins (i.e., fluorescence) and the biochemical ties of the target protein This article describes a method used to generateEscherichia coli strains with all their RNA polymerase (RNAP) moleculeslabeled with GFP, CFP, or YFP It also describes the physical and bio-chemical properties of purified RNAP molecules tagged with differentfluorescent proteins

proper-Construction of E coli Strains with Chromosomal Gene Fusions to therpoC Gene

Escherichia coli core RNAP is composed of four subunits, 20 The and 0subunits are respectively encoded by the rpoB and rpoC genes in thesame operon We fused gfp, cfp, or yfp genes at the 30end terminus of therpoC gene in the chromosome The choice of fusing the C terminus of the 0

subunit with a fluorescent protein was based on the previous finding thatthe C terminus of the 0subunit can be fused to a 200 amino acid fragment

of the galactosidase without loss of 0 function.4 A schematic of theconstruction of gene fusions is represented inFig 1

1 R Y Tsien, Annu Rev Biochem 67, 509 (1998).

2 M Chalfie and S Kain (eds.), ‘‘Green Fluorescent Protein, Properties, Applications and Protocols.’’ Wiley-Liss, New York, 1998.

3 M C Southward and M Surette, Mol Microbiol 45, 1191 (2002).

4 G C Rowland and R E Glass, Mol Microbiol 17, 401 (1995).

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PCR product

E coli chromosome rpoC

gfpuv cfp yfp

GFPuv CFP YFP

Oligo 1

Oligo 2

pGFPuvpDJ2701pDJ2702

PCR with Oligos 1 and 2

Recombination into E coli chromosome

Fig 1 A schematic for the construction of chromosomal gene fusions to the rpoC gene Regions representing sequences of the E coli chromosome and sequences with homology to it are shaded in gray The drawing is not to scale (A) Plasmid DNA templates and relative orientation of the oligonucleotides used in the PCR reaction (B) Relative location of the

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Generating Linear DNA Fragments That Carry gfpuv, cfp, or yfp Genesand the bla Gene Flanked by Chromosomal Sequences Near the End

of rpoC

We used the polymerase chain reaction (PCR) to generate linear ments to be used for recombination in the next step The DNA templatesfor these PCR reactions are pGFPuv (Clontech), pDJ2701 (which harborsthe cfp gene), or pDJ2702 (which harbors the yfp gene) Note The GFPuvprotein is a mutant GFP optimized for higher bacterial expression andmaximal fluorescence when excited by UV light The plasmids pDJ2701and pDJ2702 are pGFPuv derivatives They have been constructed by re-placing the gfpuv gene by the cfp or yfp genes The cfp and yfp genes wereobtained from the pDH3 and pDH5 plasmids (University of Washington,Seattle, Yeast Resource Center),5respectively A schematic map of theseplasmids is shown inFig 1A The relative positions of the two oligos nucle-otides (oligo 1 and oligo 2) for these PCR reactions are also shown inFig.1A The sequence of oligo 1 is 50CCAy GCC TGG CAG AAC TGC TGAACG CAG GTC TGG GCG GTT CTG ATA ACG AGC TAG AAA

GCG GGT TTT TAC GTT ATT TGC GGA TTA TGG TCT GACAGT TAC CAA TGC 30 For oligo 1, the first 50 nucleotides (nt) of the

50end (underlined) are identical to the last 50 nt coding sequence of rpoCimmediately before the stop codon, followed by an 9 nt (bold) encoding for

a three amino acid linker, Leu-Glu-Ile, that replaces the stop codon of therpoC gene Downstream of the three amino acid linker, the oligo containssequences encoding the first 29 nt coding sequences of the gfpuv, cfp, or yfpgenes (the 50end sequences of the three genes are the same) For oligo 2,the first 51 nt of the 50end (underlined) are identical to the bases located

4 bp downstream from the stop codon of the rpoC gene, and the remaining

21 nt are identical to the last coding sequences, including the stop codon

of the bla gene encoding ampicillin resistance (Ampr) The expectedPCR products are DNA fragments of 2230 bp The DNA fragments

homolog regions between the linear DNA fragment and the chromosomal rpoC gene The 50end of the linear DNA fragment, represented as a solid gray bar ( & ), is homolog to the 50 bp upstream of the stop codon of the rpoC gene The 3 0 end of the linear DNA fragment, represented as a gray wavy line ( ), is homolog to sequences located 4 bp downstream

of the rpoC gene stop codon (C) Gene fusion at the rpoC locus A linker of three codons (Leu-Glu-Ile) replaces the stop codon and fuses the rpoC gene to genes encoding the fluorescent proteins.

5 D W Hailey, T N Davis, and E G Muller, Methods Enzymol 351, 34 (2002).

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are purified The overall homology of this DNA fragment relative to theend of the rpoC gene in the chromosome is shown inFig 1B.

Recombining the Linear DNA Fragment into the E coli Chromosome

We used the recombination method described by Yu and ators,6which is particularly useful for this purpose, and selected the recom-binant candidates on LB þ Amp plates (Fig 1C) The Ampr coloniesobtained are checked to ensure they are recombinants because minuteamounts of intact plasmid templates could be present in the linear DNApreparation and thereby transform into the recipient cells, resulting inAmprcolonies A rapid method used to check this is to analyze total pro-teins from the Amprcolonies by Western blot followed by immunostainingwith either an anti-GFP antibody or a monoclonal anti-0 antibody Pro-teins from recombinant Ampr cells show a reactive band corresponding

collabor-to a fusion protein with a molecular weight higher than that of the 0peptide To confirm that the fusion is correct, we sequenced the rpoCregions flanking the insertion points, including the gfpuv, cfp, or yfp genes.rpoC–gfpuv and other fusions can be transferred to other strains’ back-grounds by phage P1 transduction using the bla gene (Ampr) as a selectionmarker (the linkage between rpoC fusion and bla is almost 100%) Note.Cells expressing 0fusion proteins have a reduced ability to grow at tem-peratures higher than 37 This defect is probably caused by a misfolding

poly-of the fluorescent proteins, which alters the conformation poly-of the entirefusion protein at temperature higher than 37.7–9Thus, strains containingrpoC fusions should be grown at 32

Purification of RNAP Tagged with Green, Yellow, or Cyan

RNAP fused with different fluorescent proteins are purified essentially

as described for the purification of RNAP.10The only modification is thatbefore Mono Q chromatography, the ammonium sulfate precipitates ofRNAP fusion proteins are resuspended in 0.2 M NaCl in TGED instead

of TGED only This is done because the NaCl apparently facilitates the

6 D Yu, H M Ellis, E C Lee, N A Jenkins, N G Copeland, and D L Court, Proc Natl Acad Sci USA 97, 5978 (2000).

7 P A Levin, I G Kurtser, and A D Grossman, Proc Natl Acad Sci USA 96, 9642 (1999).

8 K R Siemering, R Golbick, R Sever, and J Haseloff, Curr Biol 6, 1653 (1996).

9 G S Gordon, D Sitnikov, C D Webb, A Teleman, A Straight, R Losick, A W Murray, and A Wright, Cell 90, 1113 (1997).

10 M V Sukhodolets and D J Jin, J Biol Chem 273, 7018 (1998).

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solubilization of RNAP fluorescent fusion proteins It has been reportedthat the GFP dimerizes in solutions of ionic strengths less than

100 mM11; it is possible that at a very low ionic strength the highly trated RNAP fluorescent fusion proteins become insoluble A typical yieldfor core and holo RNAP fusion proteins is about 15 mg from about 100 g

concen-of wet cell paste, similar to that concen-of RNAP preparations When purified,each RNAP fusion protein has the characteristic color of the correspondingfluorescent protein.Figure 2shows a SDS–PAGE analysis of purified wild-type and different RNAP fluorescent fusion proteins As expected, 0sub-units from RNAP fusion proteins, due to fused fluorescent proteins, exhibit

a higher molecular weight than that of wild-type RNAP

Physical and Biochemical Properties of RNAP Tagged with Green,Yellow, or Cyan Fluorescent Proteins

We studied purified RNAP fusion proteins by fluorescence copy Each tagged RNAP has emission and excitation spectra that are co-incident with spectra of the corresponding isolated fluorescent protein.1

spectros-11 F Yang, L G Moss, and G N Phillips, Nature Biotechnol 14, 1246 (1996).

Fig 2 SDS–PAGE of wild-type (WT) RNAP and RNAP tagged with green, yellow, or cyan fluorescent proteins Positions of the bands corresponding to the different subunits are indicated.

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For example, peak values in excitation and emission spectra of the RNAP–GFPuv protein fusion are 396 and 507 nm, respectively (Fig 3A) Peakvalues in excitation spectra of the RNAP–CFP protein fusion are between

436 and 450 nm, whereas the peak value in emission spectra of the fusionprotein is 474 nm (Fig 3B) Peak values in excitation and emission spectra

of the RNAP–YFP protein fusion are 517 and 530 nm, respectively (Fig 3C).RNAP fluorescent fusion proteins are fully active in RNA synthesis.The fact that cells carrying chromosomal fusions between the rpoC gene

Fig 3 Excitation (dotted line) and emission spectra (continuous line) of RNAP enzymes labeled with green, yellow, or cyan fluorescent proteins Data were collected with a Quanta Master fluorometer (Photon Technology International).

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and the genes encoding fluorescent proteins are viable demonstrates thatRNAP fusion proteins are functional in vivo In vitro, transcriptional activ-ities of purified RNAP fluorescent fusion proteins are comparable to those

of the purified wild-type RNAP (Fig 4) Furthermore, RNAP fluorescentfusion proteins are as active as the wild-type RNAP in in vitro transcriptionassays at 37and 42(data not shown) These results are consistent with thenotion that the folding step of fluorescent proteins, rather than the function

of RNAP fluorescent fusion proteins, is temperature sensitive in vivo.7–9

Potential Uses of RNAP Tagged with Green, Yellow, or Cyan

RNAP fused with fluorescent proteins will provide a powerful newtool to study RNAP functions and transcriptions both in vivo and in vitro.The following examples are potential uses of RNAP fusion proteins Invivo, the distribution of RNAP inside cells under different physiological

Fig 4 In vitro transcription assays with wild-type RNAP or RNAP tagged with green, yellow, or cyan fluorescent proteins In vitro transcription reactions were carried out as described previously12in a transcription buffer containing 50 mM KCl The DNA template was plasmid pDJ631 containing the tac promoter, and RNAs were labeled with (-32P) UTP Reactions were analyzed in an 8% sequencing gel An autoradiograph of the gel is shown Positions of transcripts synthesized from tac and RNAI promoters are indicated.

12 Y N Zhou and D J Jin, Proc Natl Acad Sci USA 95, 2908 (1998).

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conditions could be visualized using a fluorescence microscope.13Also, thelocations of RNAP and another transcription factor(s) could be addressedsimultaneously by imaging cells expressing both RNAP tagged with CFP (orYFP) and a transcription factor(s) tagged with YFP (or CFP), respectively.Many transcription factors are interesting candidates for these experiments:the factors, RapA, NusA, and SspA, just to mention a few In addition,analyses of fluorescence resonance energy transfer (FRET) between thetagged RNAP and a tagged transcription factor could be used to analyzethe physical interaction between RNAP and the transcription factor.

In vitro, the movements of single RNAP molecules could be followed

or visualized under different transcription conditions using these RNAPfluorescent fusion proteins In addition, the FRET experiments mentionedearlier could also be used to study the interactions of RNAP and anothertranscription factor(s) and to follow the association and/or dissociation of

a different transcription factor(s) during the transcription cycle underdefined conditions

Acknowledgments

We thank Drs Wenxue Yang and Huijun Zhi for their assistance in the purification of RNAPs.

[2] Purification of Bacillus subtilis RNA Polymerase

and Associated Factors

By John D HelmannIntroduction

Bacterial RNA polymerase (RNAP) has been purified using a wide iety of techniques In addition to continuing interest in its fundamental en-zymatic properties, purified RNAP is used as a tool for the investigation oftranscriptional control mechanisms and for the preparation of specificRNA transcripts This article focuses on techniques developed for the puri-fication of RNAP and its associated factors from the model gram-positivebacterium Bacillus subtilis

var-Bacillus subtilis RNAP has a complex subunit structure The minimalcatalytic moiety, consisting of the 02complex, is associated with two !

13 J E Cabrera and D J Jin, Mol Microbiol (2003, in press).

Tiêu đề	RNA Polymerase and Associated Factors, Part C
Tác giả	Sankar Adhya, Susan Garges
Trường học	California Institute of Technology
Chuyên ngành	Biology
Thể loại	essay
Thành phố	Pasadena

Định dạng
Số trang	841
Dung lượng	9,8 MB