Genetics and Molecular Biology

[Pages:715]Genetics and Molecular Biology

Genetics and Molecular Biology

SECOND EDITION

Robert Schleif

Department of Biology The Johns Hopkins University Baltimore, Maryland

The Johns Hopkins University Press Baltimore and London

1986 by Addison-Wesley Publishing Company 1993 by Robert Schleif All rights reserved Printed in the United States of America on acid-free paper

The Johns Hopkins University Press 2715 North Charles Street Baltimore, Maryland 21218-4319 The Johns Hopkins Press Ltd., London

Library of Congress Cataloging-in-Publication Data

Schleif, Robert F.

Genetics and molecular biology / by Robert Schleif.--2nd ed.

p.

cm.

Includes bibliographical references and index.

ISBN 0-8018-4673-0 (acid-free paper).--ISBN 0-8018-4674-9 (pbk : acid-free

paper)

1. Molecular genetics. I. Title

QH442.S34 1993

The catalog record for this book is available from the British Library.

Preface

This book evolved from a course in molecular biology which I have been teaching primarily to graduate students for the past twenty years. Because the subject is now mature, it is possible to present the material by covering the principles and encouraging students to learn how to apply them. Such an approach is particularly efficient as the subject of molecular genetics now is far too advanced, large, and complex for much value to come from attempting to cover the material in an encyclopedia-like fashion or teaching the definitions of the relevant words in a dictionary-like approach. Only the core of molecular genetics can be covered by the present approach. Most of the remainder of the vast subject however, is a logical extension of the ideas and principles presented here. One consequence of the principles and analysis approach taken here is that the material is not easy. Thinking and learning to reason from the fundamentals require serious effort, but ultimately, are more efficient and more rewarding than mere memorization.

An auxiliary objective of this presentation is to help students develop an appreciation for elegant and beautiful experiments. A substantial number of such experiments are explained in the text, and the cited papers contain many more.

The book contains three types of information. The main part of each chapter is the text. Following each chapter are references and problems. References are arranged by topic, and one topic is "Suggested Readings". The additional references cited permit a student or researcher to find many of the fundamental papers on a topic. Some of these are on topics not directly covered in the text. Because solving problems helps focus one's attention and stimulates understanding, many thought-provoking problems or paradoxes are provided. Some of these require use of material in addition to the text. Solutions are provided to about half of the problems.

v

vi Preface

Although the ideal preparation for taking the course and using the book would be the completion of preliminary courses in biochemistry, molecular biology, cell biology, and physical chemistry, few students have such a background. Most commonly, only one or two of the above-mentioned courses have been taken, with some students coming from a more physical or chemical background, and other students coming from a more biological background.

My course consists of two lectures and one discussion session per week, with most chapters being covered in one lecture. The lectures often summarize material of a chapter and then discuss in depth a recent paper that extends the material of the chapter. Additional readings of original research papers are an important part of the course for graduate students, and typically such two papers are assigned per lecture. Normally, two problems from the ends of the chapters are assigned per lecture.

Many of the ideas presented in the book have been sharpened by my frequent discussions with Pieter Wensink, and I thank him for this. I thank my editors, James Funston for guidance on the first edition and Yale Altman and Richard O'Grady for ensuring the viability of the second edition. I also thank members of my laboratory and the following who read and commented on portions of the manuscript: Karen Beemon, Howard Berg, Don Brown, Victor Corces, Jeff Corden, David Draper, Mike Edidin, Bert Ely, Richard Gourse, Ed Hedgecock, Roger Hendrix, Jay Hirsh, Andy Hoyt, Amar Klar, Ed Lattman, Roger McMacken, Howard Nash, and Peter Privalov.

Contents

1 An Overview of Cell Structure and Function

1

Cell's Need for Immense Amounts of Information

2

Rudiments of Prokaryotic Cell Structure

2

Rudiments of Eukaryotic Cell Structure

5

Packing DNA into Cells

7

Moving Molecules into or out of Cells

8

Diffusion within the Small Volume of a Cell

13

Exponentially Growing Populations

14

Composition Change in Growing Cells

15

Age Distribution in Populations of Growing Cells

15

Problems

16

References

18

2 Nucleic Acid and Chromosome Structure

21

The Regular Backbone Of DNA

22

Grooves in DNA and Helical Forms of DNA

23

Dissociation and Reassociation of Base-paired Strands

26

Reading Sequence Without Dissociating Strands

27

Electrophoretic Fragment Separation

28

Bent DNA Sequences

29

Measurement of Helical Pitch

31

Topological Considerations in DNA Structure

32

Generating DNA with Superhelical Turns

33

Measuring Superhelical Turns

34

Determining Lk, Tw, and Wr in Hypothetical Structures

36

Altering Linking Number

37

Biological Significance of Superhelical Turns

39

vii

viii Contents

The Linking Number Paradox of Nucleosomes

40

General Chromosome Structure

41

Southern Transfers to Locate Nucleosomes on Genes

41

ARS Elements, Centromeres, and Telomeres

43

Problems

44

References

47

3 DNA Synthesis

53

A. Enzymology

54

Proofreading, Okazaki Fragments, and DNA Ligase

54

Detection and Basic Properties of DNA Polymerases

57

In vitro DNA Replication

60

Error and Damage Correction

62

B. Physiological Aspects

66

DNA Replication Areas In Chromosomes

66

Bidirectional Replication from E. coli Origins

67

The DNA Elongation Rate

69

Constancy of the E. coli DNA Elongation Rate

71

Regulating Initiations

72

Gel Electrophoresis Assay of Eukaryotic Replication Origins

74

How Fast Could DNA Be Replicated?

76

Problems

78

References

79

4 RNA Polymerase and RNA Initiation

85

Measuring the Activity of RNA Polymerase

86

Concentration of Free RNA Polymerase in Cells

89

The RNA Polymerase in Escherichia coli

90

Three RNA Polymerases in Eukaryotic Cells

91

Multiple but Related Subunits in Polymerases

92

Multiple Sigma Subunits

95

The Structure of Promoters

96

Enhancers

99

Enhancer-Binding Proteins

100

DNA Looping in Regulating Promoter Activities

102

Steps of the Initiation Process

104

Measurement of Binding and Initiation Rates

105

Relating Abortive Initiations to Binding and Initiating

107

Roles of Auxiliary Transcription Factors

109

Melted DNA Under RNA Polymerase

110

Problems

111

References

113

5 Transcription, Termination, and RNA Processing

119

Polymerase Elongation Rate

119

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