Chapter category: Gene Expression
Transcription
RNA Polymerase III Transcription
Second Edition
Edited by: Robert J. WhiteISBN: 1-57059-482-1
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Chapter authors:
Robert J. White
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Once a preinitiation complex has formed on a yeast tRNA gene, RNA chain ini-tiation requires a further 5 min at 22°C (half-life ~2 min).1 During this period, three successive steps occur: recruitment of pol III, melting of the DNA helix, and initiation of RNA synthesis. Recruitment and melting are the slow steps, whereas the subsequent synthesis of a seventeen base transcript is completed within 10 seconds.1
On its assembly into a preinitiation complex, yeast pol III generates a footprint from –3 to +21 on the SUP4 tRNATyr gene and from –10 to +13 on the 5S RNA gene.2 A weak sequence homology between various class III genes occurs at the initiation site and mutations in this region can reduce transcription strongly, in some instances.3–8 Since the homology lies within the footprint of pol III, it may be recognized directly by the polymerase itself. Six different subunits of yeast pol III can be crosslinked specifically to DNA within the assembled preinitiation complex.9,10 The C160 and C128 subunits are sufficiently extended along the DNA to be accessible to photoreactive residues situated anywhere between –17 and +20, while C34 extends from –21 to +6.9 Its projection from the trailing edge of the polymerase allows C34 to interact with TFIIIB. In contrast, ABC27 is situated at the leading edge of the polymerase before initiation begins.9 C82, C53, C31, and AC40 or C37 (these polypeptides could not be clearly resolved) can all be crosslinked to DNA at more restricted and centrally placed positions within the preinitiation complex.9
Pol III "melts" the DNA at the start site in a process that can be monitored by footprinting with potassium permanganate.2,6 Permanganate oxidizes thymine in single-stranded DNA but has little effect upon thymine in duplex DNA.11 Permanganate probing experiments indicate that prior to initiation pol III induces a reversible change in the conformation of the DNA due to opening of the helix.2,6 The melted region at the SUP4 tRNA promoter extends from –11 to +11.6 The A/T-rich sequences that tend to flank transcription start sites may facilitate promoter opening due to the lower thermodynamic stability of A:T base pairs. Although pol III can bind to the preinitiation complex at 0°C, the process of template melting is highly temperature-dependent and increases progressively from 10°C to 40°C, with a sharp transition between 10°C and 15°C.6 The extent of DNA opening and its temperature dependence are reminiscent of the melting process that occurs at bacterial promoters.12 However, whereas promoter bound E. coli RNA polymerase is in rapid equilibrium with free enzyme as long as the template remains closed, yeast pol III associates stably with the preinitiation complex even prior to promoter melting.6
Additional chapters from this book:
Perspective
Robert J. White
Our understanding of the details of pol III transcription has increased substan-tially in recent years. The characterization and cloning of many of the components of the system have been ac...
Regulation of RNA Polymerase III Transcription
Robert J. White
There are two families of active 5S genes in Xenopus laevis. One consists of the somatic 5S genes, of which there are 400 copies per haploid genome, organized in a single cluster.
Proteins that Modulate the Rate of RNA Polymerase III Transcription
Robert J. White
So far I have described the basal pol III transcription apparatus and how this functions to allow expression of class III genes. The level of transcription can be modulated in either a posi...
Chromatin Structure of Class III Genes
Robert J. White
The chromatin structure of a gene can be a major determinant of its transcrip-tional activity (reviewed in refs. 1–8). In chromatin, 146 bp of DNA is wrapped approximately twice around...
Transcription
Robert J. White
Once a preinitiation complex has formed on a yeast tRNA gene, RNA chain ini-tiation requires a further 5 min at 22°C (half-life ~2 min).1 During this period, three successiv...
Transcription Complex Formation on Class III Genes
Robert J. White
The formation of transcription complexes, composed of factors bound to DNA, was initially investigated by means of the template exclusion assay. This approach monitors the ability of a gene...
Transcription Factors Utilized by RNA Polymerase III
Robert J. White
Purified pol III initiates transcription randomly.1–4 Accurate and specific initia-tion requires the assistance of transcription factors in order to recruit the polymerase t...
RNA Polymerase III
Robert J. White
Pol III is the largest of the nuclear RNA polymerases, with an aggregate molecular weight of 600700 kD (reviewed by Thuriaux and Sentenac1–3). This is, perhaps, surprising s...
Promoter Structure of Class III Genes
Robert J. White
The promoters of most class III genes include discontinuous intragenic structures, termed internal control regions (ICRs), that are composed of essential sequence blocks separated by noness...
Class III Genes
Robert J. White
The genes transcribed by pol III encode a variety of small RNA molecules. (Table 1) Many of these have essential functions in cellular metabolism, such as tRNA and 5S rRNA, which are required ...
