The Ribonucleic Acids

279 Pages·1973·6.82 MB·English

by  D. S. Letham

The Ribonucleic Acids
Edited by
P. R. Stewart and D. S. Letham
With Contributions by
G. D. Clark-Walker· L. Dalgarno· A. J. Gibbs
A. J. Howells· D. S. Letham· H. Naora
G. M. Polya . R. Poulson· J. Shine· J. J. Skehel
P. R. Stewart· P. R. Whitfeld
With 56 Figures
Springer-Verlag
Berlin· Heidelberg· New York 1973
ISBN 978-3-662-00501-9 ISBN 978-3-662-00499-9 (eBook)
DOI 10.1007/978-3-662-00499-9
This work is subject to copyright. All rights are reserved, whether the whole or part of the
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© by Springer-Verlag Berlin' Heidelberg 1973. Library of Congress Catalog Card Number
73-76335.
Saftcover reprint afthe hardcaver 1st edition 1973
Foreword
The central role of the ribonucleic acids (RNA) in mediating the
expression of information encoded in DNA in living cells is now
well established. Research in this area of biology continues at a
remarkable rate, and new and significant information appears
almost daily in a wide range of journals, published symposia and
specialist reviews.
The scattered nature of this information makes it difficult for
the newcomer to the field of ribonucleic acid biochemistry to
obtain a general oversight of current activity and new advances.
Moreover, the reviews available for the most part are concerned
with rather insular aspects of these ubiquitous molecules, or in the
case of text-books, the subject is treated as part of a general out
line of properties of nucleic acids and thus often tends to be
superficial.
With these considerations in mind, a postgraduate course was
instituted in the university in Canberra to attempt to provide a
comprehensive, though not excessively detailed, outline of the
biological roles of RNA. The course was designed for students
with a sound undergraduate training in biochemistry, but other
wise with a wide variety of biological interests-plant physiology,
virology, organelle biochemistry, genetics.
The chapters in this book represent the matter of that course.
The collection is not meant to be a completely comprehensive
account of the latest findings in the field. The book is aimed at
those already initiated in the basic biology and chemistry of RNA,
and who are probably pursuing some aspect of cellular develop
ment and differentiation. Hopefully, it provides a grounding in
established facts and concepts in most aspects of the function of
RNA species. Newer developments that seem to be important and
are likely to result in a greater understanding of the role of RNA
in living cells are also dealt with.
Canberra, January 1973 P. R. STEWART
D. S. LETHAM
Contents
CHAPTER 1
RNA in Retrospect. By D. S. LETHAM, P. R. STEWART, and
G. D. CLARK-WALKER. 1
References. . . . . . . . . . . . . . . . . . . .. 5
CHAPTER 2
Transcription. By G. M. POLYA . . . . . . . 7
Introduction . . . . . . . . . . . . . . 7
A. Purification and Physical Properties of RNA Poly-
merases. . . . . . . . . . . . . . 7
1. General Procedural Considerations . 7
2. Purification of RNA Polymerases . . 9
3. Physical Properties of RNA Polymerases 11
B. Initiation of Transcription. . . . . . . . 12
1. Non-Specific Binding of RNA Polymerase to DNA. 13
2. Specific RNA Polymerase Binding to Initiation
Sites. . . . . . . . . . . . . . . . . . . . . 13
3. DNA Strand Separation as a Consequence of RNA
Polymerase Binding . . . . . . . . . . . . . . 14
4. Formation of a Rifampicin-Resistant Preinitiation
Complex. . . . . . . . . . . . . . . . 15
5. Binding of the First Nucleotide . . . . . . 15
6. Formation of the First Phospho diester Bond 15
C. Elongation and Termination of RNA Chains 16
1. Elongation of RNA Chains. . . . . . . . 16
2. Termination and Reinitiation of RNA Chains . 17
D. Inhibitors of RNA Polymerase . . . . . . 17
1. DNA-Specific Inhibitors of Transcription. . 17
2. Protein-Specific Inhibitors of Transcription . 22
3. Polyanions as Inhibitors of RNA Polymerase 24
4. Nucleotide Analogues as Inhibitors of RNA Poly-
merase. . . . . . . . . . . . . . . . . 24
5. Physiological Inhibitors of RNA Polymerase 24
E. Control of Prokaryote Transcription 24
1. Repressors . . . . . . . . . . . . . . . 25
VIII Contents
2. Specific Transcription Activation Factors . 25
3. Control of Bacteriophage Transcription 28
F. Control of Eukaryote Transcription . . 29
1. Specific Inhibitors of rRNA Synthesis . 29
2. Transcription Activation Factors . . . 29
3. The Role of Chromosomal RNA and Histones in
Gene Repression . . . . . . . . . . . . . . . 30
4. Acidic Nuclear Proteins and Cyclic AMP Control
of Transcription . . . . . . . . . . . . . . . 30
5. Effects of Hormones on Higher Plant Transcription 31
6. Effects of Hormones on Transcription in Animal
Systems 31
References. . . . . . . . . . . . . . . . . . . . . 32
CHAPTER 3
Nuclear RNA. By H. NAORA 37
Introduction . . . . . . 37
A. Preparation of Nuclear RNA 37
1. Isolation of Nuclei. . . . 37
2. Preparation of RNA from Isolated Nuclei 38
3. Differential Extraction of Cellular RNA . 38
B. The Concept of Nucleus-Specific RNA and Migrat-
ingRNA .............. . 39
C. Heterogeneous Nuclear RNA (HnRNA) . . . . . 40
1. Synthesis and Degradation of HnRNA. . . . . 40
2. Is HnRNA a Precursor of Cytoplasmic mRNA? . 41
3. Some Comments on HnRNA. . . . . . . 45
D. Low Molecular Weight Nuclear RNA (LnRNA) 46
1. Physical and Chemical Properties 47
2. Intracellular Distribution. . . . 49
3. LnRNA-Nucleoprotein Particles. 49
4. Synthesis and Metabolic Stability 50
5. The Relationship between LnRNA and Other RNA
Species .......... . 50
6. Possible Functions of LnRNA . . . . . . . 51
E. Chromosomal RNA (ChRNA) . . . . . . . . 52
1. The Properties of ChRNA as Described by the
Bonner and Huang Groups . 53
2. The Critical View of ChRNA 54
References . 55
CHAPTER 4
Messenger RNA. By A. J. HOWELLS . . . . . . . . . 59
Introduction . . . . . . . . . . . . . . . . . . 59
A. Nucleotide Sequencing Studies on Bacteriophage
mRNA .................. 59
Contents IX
1. The Single-Stranded RNA Viruses of E. coli . 59
2. Techniques for Sequence Analysis . . 60
3. The Biochemical Map of R 17 RNA. . 61
4. Possible Secondary Structure . . . . . 65
5. Natural Degeneracy of the Genetic Code 67
6. Summary of Studies of Bacteriophage mRNA . 68
B. Purification of Non-Viral mRNA Species and Prelimi
nary Sequencing Studies . . . . . . . . . . . . . 69
1. The Determination and Characterization of mRNA 69
2. The Isolation of Globin mRNA. . . . . . . . . 71
3. Characterization of Globin mRNA . . . . . . . 73
4. Nucleotide Sequencing Studies with Globin mRNA 75
5. The Purification of Other Species of mRNA 75
6. The Stability of Non-Viral Species of mRNA 77
References. . . . . . . . . . . . . . . . . . 78
CHAPTER 5
Transfer RNA and Cytokinins. By D. S. LETHAM . 81
Introduction . . . . . . . . . . . . . . 81
A. Isolation of Amino Acid-Specific tRNA 81
B. The Structure of tRNA. . . . . . . . 83
1. Sequence Determination; Primary and Secondary
Structures of Yeast Alanine tRNA and Certain Iso-
accepting tRNAs . . . . . . . . . . . . .. 83
2. Homology in Primary and Secondary Structures . 87
3. Tertiary Structure. . . . . . . . . . . . .. 90
C. Biosynthesis of tRNA . . . . . . . . . . . .. 93
D. The Aminoacylation of tRNA; the Role of Aminoacyl-
tRNA in Biosynthesis . . . . . . 95
E. tRNA and Regulatory Mechanisms. . . . . 96
1. Translation. . . . . . . . . . . . . . 96
2. End Product Repression; Enzyme Activity 99
3. Cytokinins and tRNA 100
References. . . . . . . . . . . . . . . . . 104
CHAPTER 6
Ribosomal RNA. By L. DALGARNO and J. SHINE. 107
Introduction . . . . . . . . . . . 107
A. The Structure of Ribosomal RNA 107
1. Molecular Weight. . . . . . 107
2. Base Composit:on. . . . . . 108
3. Base Sequence of High Molecular Weight rRNA 109
4. Base Sequence of Low Molecular Weight rRNA. 114
5. Heterogeneity in the Base Sequence of rRNA 114
6. The Existence of 'Hidden Breaks' in rRNA . 115
7. Secondary Structure of rRNA. . . . . . . 116
x Contents
B. The Synthesis of Ribosomal RNA 120
1. Eukaryotes . 120
2. Prokaryotes 129
References . 132
CHAPTER 7
Translation of Messenger RNA. By G. D. CLARK-WALKER. 135
Introduction . . . . . . . . 135
A. Initiation. . . . . . . . . 136
1. Formylmethionyl-tRNA . 136
2. Protein Initiation Factors 136
3. Steps in Initiation . . . . 136
4. Regulation of Translation by Initiation Factors 137
5. Initiation in Eukaryotic Organisms 138
B. Elongation . . . . . . . . . . . . 139
1. Binding . . . . . . . . . . . . 139
2. Linkage (Peptide Bond Formation) 141
3. Translocation. . . . . . . . . . 141
4. Elongation in Eukaryotic Organisms. 142
C. Termination. . . . . . . . . . . . 143
D. The Ribosome Cycle. . . . . . . . . 144
E. Translation of Polycistronic Messenger . 144
1. Occurrence of Polycistronic Messenger and Its Trans-
lation . . . . . . . . . . . . . 144
2. Polarity . . . . . . . . . . . . 146
F. Antibiotics as Inhibitors of Translation 146
G. Concluding Remarks. 146
References. . . . . . . . . . . . . . 147
CHAPTER 7: Appendix
Inhibitors of Translation. By P. R. STEWART. 151
Introduction . . . . . . . . . . . . . 151
A. General Properties of Translation Inhibitors . 151
B. Summary of Primary Effects in vitro . . 155
1. Inhibitors of Small Subunit Function 155
2. Inhibitors of Large Subunit Function. 156
C. Inhibitors of Organelle Protein Synthesis 157
References. . . . . . . . . . . . . . . 158
CHAPTER 8
Mitochondrial RNA. By P. R. STEWART ......... . 159
Introduction . . . . . . . . . . . . . . . . . . . . 159
A. Experimental Problems Associated with the Isolation
and Characterization of RNA from Mitochondria 159
B. Mitochondrial Ribosomes 161
Contents XI
1. Location within the Organelle. . . . . . . . . . 161
2. Physical and Chemical Properties . . . . . . . . 161
C. Ribosomal RNA of Mitochondria from Yeast, Fungi
and Protozoa . . . . . . . . . . . . . . . . . . 162
D. Ribosomal RNA of Mitochondria from Higher Or-
ganisms . . . . . . . . . . . . . . . . 163
E. Unusual Properties of Mitochondrial rRNA . 165
F. Transfer RNA . . . . . . . . . . 167
G. The Synthesis of Mitochondrial RNA 167
1. Genes for Mitochondrial RNA . . 167
2. Transcription in Mitochondria . . 171
3. Synthesis of Mitochondrial Ribosomal Proteins 172
4. Mitochondrial RNA and Development. 173
H. Concluding Remarks. 175
References. . . . . . . . . . . . . . . . 175
CHAPTER 9
Chloroplast RNA. By P. R. WHITFELD 179
Introduction . . . . . . . 179
A. Isolation of Chloroplasts 179
1. Nonaqueous Method 180
2. Aqueous Method . . 180
B. Extraction of Leaf and Chloroplast RNAs . 181
C. Transfer RNA . . . . . . . . . . . . 182
1. Formylmethionine tRNA . . . . . . 182
2. Other Species of tRNAs in Chloroplasts 183
3. Coding Information for Chloroplast tRNAs 184
D. Ribosomes . . . . . . . . . . . . . . . . 185
1. Properties and Distribution within the Chloroplast. 185
2. Isolation of Chloroplast Ribosomes . . . . . . . 186
E. Ribosomal RNA. . . . . . . . . . . . . . . . . 187
1. Molecular Size of Ribosomal RNA from Chloro-
plasts . . . . . . . . . . . . . . . . . .. 187
2. Isolation of Chloroplast Ribosomal RNA 188
3. Characteristics of Chloroplast Ribosomal RNAs . 189
4. Synthesis of Chloroplast Ribosomal RNA . .. 193
5. Coding Information for Chloroplast Ribosomal
RNA. . . . . . . . . . . . 194
6. Chloroplast 5S Ribosomal RNA. . . 196
F. Messenger RNA (mRNA). . . . . . . 196
G. RNA Synthesis in Isolated Chloroplasts. 198
1. General Features . . . . . . 198
2. Product of the Reaction . . . . . . 199
3. Chloroplast RNA Polymerase. . . . 200
H. RNA Synthesis in Developing Chloroplasts 201
XII Contents
I. Concluding Remarks . 203
References. . . . . . . 204
CHAPTER 10
Viral RNA. By A. J. GIBBS and J. J. SKEHEL 207
Introduction . . . . . . . . . . . . 207
A. Synthesis and Function . . . . . . 208
1. Viruses Whose Particles Contain Single Stranded In
fectious RNA. . . . . . . . . . . . . . . . . 208
2. Viruses Whose Particles Contain Single Stranded
Noninfectious RNA .............. 215
3. Viruses Whose Particles Contain Double Stranded
RNA. . . . . . . . . . . . . . . 219
4. Viruses Whose Particles Contain DNA. 219
B. Structure of Viral RNAs . . . . . . . 223
1. RNAs Extracted from Virus Particles 223
2. RNA within Virus Particles. . . . . 228
3. Particle Reassembly Experiments . . 229
4. Viral RNAs Not Found in Virus Particles. 231
C. Concluding Remarks . 232
Glossary 232
References. . . . . . . 239
CHAPTER 11
Isolation, Purification and Fractionation of RNA
By R. POULSON. . . 243
Introduction . . . . . . 243
A. Tissue Disruption . . 243
1. Extraction Medium 243
2. Disruption of Cells and Organelles. 245
B. Nucleoprotein Dissociation and Deproteinization of
Released RNA 247
1. Materials. 247
2. Methods. . . 248
C. Purification. . . 253
1. Separation of RNA from Polysaccharides. 253
2. Separation of rRNA from tRNA and DNA. 254
3. Separation of tRNA from rRNA and DNA. 255
D. Fractionation . . . . . . . . . . . . . . . 256
1. Zone Velocity Centrifugation in Sucrose Gradients. 256
2. Methylated Albumin Kieselguhr (MAK) Chromato-
graphy. . . . . . . . . . . . . 257
3. Polyacrylamide Gel Electrophoresis 257
References . 259
Subject Index . . . . . . . . . . . . . . 263