Eukaryotic Gene Regulation

Volume II

Gerald M. Kolodny

• Publisher: CRC Press
• Year: 2018
• ISBN:
  9781351088824
• Edition: 1
• Topic: Medicine
• Subtopic: Alternative & Complementary Medicine

The cause of cancer and its many manifestations is at present unknown. Since many of its manifestations, including is control of cell division, appear to represent abnormal patterns of gene expression, studies of the regulation of gene expression nwill provide important insights in the understanding and treatment of cancer. This volume attempts to present some of the recent work on regulation of gene expression in eukaryotic cells.

Chapter 1

Eukaryotic Dna-Dependent Rna Polymerases: An Evaluation of Their Role in the Regulation of Gene Expression

Trevor J. C. Beebee and Peter H. W. Butterworth

I. Control of Gene Expression at the Level of the RNA Polymerase

A. Prokaryotic Precedents

B. The Eukaryotic Problem

II. The Status of the DNA-Dependent RNA Polymerase in Nucleated Cells

A. Isolation and Purification of Eukaryotic RNA Polymerases

B. Localization of RNA Polymerase Activities in Eukaryotic Cells

C. Structure and Microheterogeneity of Eukaryotic RNA Polymerases

1. Class I RNA Polymerase

2. Class II RNA Polymerase

3. Class III RNA Polymerase

D. The Function of Each Class of Eukaryotic RNA Polymerase

E. Selective Transcription by Eukaryotic RNA Polymerases In Vitro

III. Theoretical Basis for the Regulation of RNA Synthesis at the Level of the RNA Polymerases

A. Gross Alteration in the Amount of Active Enzyme

B. A Change in the Catalytic “Efficiency” of Active Polymerase

C. Modulation of RNA Polymerase Specificity

IV. Some Guidelines for the Assessment of Experimental Information

V. Systems Exhibiting Transcriptional Control

A. Development and Differentiation

B. Tumor Cells

C. Tissue Regeneration

D. Chemical Effectors

E. Hormones

F. The Stringent Response

G. Mechanisms of RNA Polymerase Alteration

1. Covalent Modification

2. Factors

3. Polyamines

VI. General Assessment of Current Knowledge

VII. Control at the RNA Polymerase Level

VIII. Future Possibilities

IX. Recent Progress

Acknowledgments

References

I. Control of Gene Expression at the Level of the RNA Polymerase

Any temptation to believe that the regulation of gene expression in nucleated cells might lie primarily at the level of transcription has its origin in the precedents established for prokaryotic systems. Simple DNA sequence (where sequences are represented in the chromosome once or only a few times), multiple structural genes often existing within one transcription unit, very rapid turnover of messenger RNA (mRNA), and concurrent transcription and translation limit the scope for regulation to occur at any point other than at the level of transcription in prokaryotes. On the other hand, eukaryotes contain amounts of DNA grossly in excess of that required to code for cellular proteins, and the DNA is extremely complex (in terms of the multiplicity of sequence repeats); a large proportion of the primary gene transcripts, constructed within the nucleus, never reaches the cytoplasm; there is a spatial separation of transcriptional from translational events which requires the transport of information from the nucleus to the site of protein synthesis. Thus, the potential for regulating the expression of specific sections of the chromosome can be dictated by any number or all of these steps in this highly organized process. With the current state of our knowledge and the limited but rapidly developing technology, one must exercise real caution in ascribing primary regulatory significance to any one of these phenomena.

This chapter concerns one stage in the overall process of expression of genetic information in eukaryotic cells: the role of DNA-dependent RNA polymerases in the controlled expression of specific (classes of) genes. The real question is whether any RNA polymerase exerts a direct regulatory influence in any system. To invoke this enzyme as being directly involved in control processes, variable initiation specificity, elongation rate, or termination efficiency must be defined in terms of some structural modification of the enzyme itself.

A. Prokaryotic Precedents

In bacterial systems, not all potential control mechanisms satisfy these strict terms of reference. In general terms, two categories of regulatory mechanism are prevalent: positive and negative control, both of which operate at the level of transcription (reviewed by Lewin¹). Both mechanisms require the binding of a control element to DNA in the proximity of the RNA polymerase binding site: a positive control element interacts with DNA at a site adjacent to and promotes the recognition of this site by the polymerase (for example, the cyclic-AMP binding protein which facilitates the binding of the polymerase to the lac promoter²); a negative control element binds to the DNA at a site which prevents the movement of the RNA polymerase from the promoter into the region containing the structural genes, e.g., the binding of repressor to the operator of the lac operon.³ Neither of these control proteins interacts directly with the RNA polymerase, and therefore, they do not satisfy the criteria for control operating directly on the enzyme.

However, there are at least three other possible control mechanisms which, under certain conditions, may be operative in bacterial cells. There is ample evidence that different RNA polymerases bind to and transcribe from different promoters; that is, they have different template specificities. Following infection by the phage T₇, the host Escherichia coli RNA polymerase transcribes a limited portion of the phage genome (the “early” genes); one of the early gene products is an RNA polymerase which recognizes the promoters for the “late” phage genes.⁴ A more subtle situation is displayed in the modification of the Bacillus subtilis RNA polymerase following infection by phage SP01;⁵ expression of viral genes results in the binding of virus-coded proteins to the host polymerase which promote the transcription of specific sections of the phage genome. Perhaps the most challenging mechanism of control which has been proposed for prokaryotes is that of Travers⁶ which suggests a role for auxiliary factors which associate with the RNA polymerase mediating the recognition of specific classes of promoters, e.g., those for ribosomal RNA coding sequences. While positive and negative control elements are involved primarily in the “fine” regulation of expression of small groups of genes under coordinate control, these alternative mechanisms dictate a “coarse” form of control, where gross changes in cellular function are to be expressed.

Although this brief survey of prokaryotic control systems begs the criticism of superficiality, it serves to place in perspective different systems of control which may be operative at the level of transcription of genetic information in bacteria.

B. The Eukaryotic Problem

We are faced with a much more complex problem when attempting to rationalize the types of mechanism regulating gene expression in nucleated cells. From a single fertilized egg, the complete organism develops through embryogenesis into a complex array of different cell phenotypes. As phenotype changes during this process, the patterns of gene expression must be changed. Ultimately, the adult phenotype is maintained through subsequent generations. It is conceivable that the “fine” contol of cellular processes may still be achieved by mechanisms similar to those operating in bacterial cells through the transient association of regulatory proteins with the chromosome. However, during replication, when associated proteins must be released from the chromosome, the opportunity arises to replace or maintain a complex array of regulatory factors which direct the expression of phenotype. This group of proteins are normally referred to as “nonhistone” proteins, whereas the histones (which form the protein core of the nucleosomal particle⁷) have a predominantly structural role in the organization and packaging of the chromosome.

If discussion is restricted to the differentiated eukaryotic cell, it is known that, in general, 50 to 80% of the genome is made up of sequences represented only once (or a few times) in the chromosome: the unique or nonrepetitive sequences. The remainder of the genome (excluding satellite DNA) is composed of “moderately repetitive” sequences (see Lewin⁸ for review). While there are some notable exceptions (such as the coding sequences for histones), most structural genes exist as single copies in the hap-loid genome. Current models of the organization of DNA sequence suggest the inter-spersion of unique with repetitive regions and invoke a regulatory function for the latter.⁹ However, the modus operandi for these putative regulatory sequences is still far from clear: it is conceivable that they constitute multiple recognition sites for the binding of RNA polymerase and/or regulatory factors (of the “positive” or “negative” control type) or sequences which, when transcribed, constitute processing enzyme cleavage points. The fact that only a small proportion of the genome (up to 10%) may be transcribed at any time confers a major role on proteins which restrict the access of the RNA polymerase to the chromosome. Almost invariably, the initial RNA transcripts are considerably longer than the sequences which ultimately appear in the cytoplasm, the latter representing sequences complementary to only about 2% of the genome.⁹ The generation of mature RNA by progressive cleavage of the primary gene transcript is, of course, a potential regulatory step. In the case of stable RNA species (rRNA and tRNA), the structural relationships and the cleavage steps required to generate mature RNAs are comparatively well characterized (extensively reviewed by Perry¹⁰), and there is some experimental evidence to suggest that control is exercised during these processes.

However, the precursor-product relationship between rapidly synthesized, high molecular weight, nuclear (HnRNA) and mRNA has been a contentious issue for more than 10 years” and has only recently received unequivocal experimental support. A high proportion of HnRNA is turned over within the nucleus. From a detailed comparison of total nuclear HnRNA and messenger populations of single cell types, HnRNA has been shown to be as much as five- to tenfold more complex than mRNA and contains repeated sequences. The evidence suggests that repetitive and nonrepeti-tive sequences are interspersed in the region of the HnRNA molecule 5′ to the mRNA component (assuming that message is located at the 3′ termini of these molecules).¹² During the maturation process, these 5′ sequences may have a role in the (controlled) selection of RNA to be transported from the nucleus to the cytoplasm; some of the repetitive sequences may form regions of secondary structure which may be recognized by processing enzymes¹³ or which link remote portions of the HnRNA molecule¹⁴ so that the sequence which appears in the cytoplasm is made up of noncontiguous sections of the primary gene transcript.¹⁵ The opportunity now exists to examine in detail the relationship between the structure of the macromolecular precursor of a specific mRNA and subsequent processing events: Bastos a...

Table of contents

• Cover
• Title Page
• Copyright Page
• Foreword
• The Editor
• Contributors
• Contents
• Chapter 1. Eukaryotic Dna-Dependent Rna Polymerases: An Evaluation of Their Role in the Regulation of Gene Expression
• Chapter 2. Role of Histones in Cell Differentiation
• Chapter 3. Role of Nonhistone Chromosomal Proteins in Selective Gene Expression
• Chapter 4. Hormonal Regulation of Gene Transcription
• Index