Genetic exploration of interactive domains in RNA polymerase II subunits.

Abstract

The two large subunits of RNA polymerase H, RPB1 and RPB2, contain regions of extensive homology to the two large subunits of Escherichia coli RNA polymerase. These homologous regions may represent separate protein domains with unique functions. We investigated whether suppressor genetics could provide evidence for interactions between specific segments of RPB1 and RPB2 in Saccharomyces cerevisiae. A plasmid shuffle method was used to screen thoroughly for mutations in RPB2 that suppress a temperature-sensitive mutation, rpbl-1, which is located in region H of RPB1. All six RPB2 mutations that suppress rpbl-1 were clustered in region I of RPB2. The location of these mutations and the observation that they were allele specific for suppression of rpbl-l suggests an interaction between region H of RPB1 and region I of RPB2. A similar experiment was done to isolate and map mutations in RPB1 that suppress a temperature-sensitive mutation, rpb2-2, which occurs in region I of RPB2. These suppressor mutations were not clustered in a particular region. Thus, fine structure suppressor genetics can provide evidence for interactions between specific segments of two proteins, but the results of this type of analysis can depend on the conditional mutation to be suppressed.Yeast RNA polymerase II is composed of 10 polypeptide subunits whose molecular masses range from 190 to 10 kilodaltons (20). The genes that encode RPB1 and RPB2, the two largest subunits, have been cloned and sequenced (2, 21). RPB1 and RPB2 show extensive sequence similarity to the Escherichia coli RNA polymerase subunits P' and P, respectively. The areas of most extensive sequence similarity between the procaryotic and eucaryotic subunits have been termed homology regions (9). RPB1 has eight regions of homology, regions A through H (2), and RPB2 has nine, regions A through I (21). The presence of these regions of conserved amino acids has led to the suggestion that they define separate protein domains, and it is possible that each of these domains has a specific function that is conserved among RNA polymerases (18).Multiple functions have been attributed to the two large subunits of procaryotic and eucaryotic RNA polymerases. The E. coli RNA polymerase has been most extensively studied in this regard (4,23 Suppression of conditional mutations is a genetic means of identifying genes whose protein products interact with the product of a mutated gene (6). Suppression genetics has been used to reveal protein-protein interactions in the large multisubunit complexes of bacterial ribosomes (14) and eucaryotic flagella (10). It has also led to the isolation of new conditional mutations in P22 bacteriophage genes that encode structural components of the phage and permitted an analysis of the path of assembly of these components (8). The a-tubulin gene of Aspergillus nidulans was isolated by suppression of a conditional ,B-tubulin mutation (13). An actin-associating protein was isolated by suppression of a conditional mutation in actin (1). Suppressors of a Salmonel...

Suppressor analysis of temperature-sensitive mutations of the largest subunit of RNA polymerase I in Saccharomyces cerevisiae: a suppressor gene encodes the second-largest subunit of RNA polymerase I.

“…Perhaps the simplest interpretation of their results and the present results is that both amino-terminal and carboxyl-terminal sequences of the largest subunit interact directly with a single region near the carboxyl terminus of the second-largest subunit. In addition, as also discussed by Martin et al (23), there remains the possibility that the genetic suppression observed in our work as well as theirs is a consequence of indirect interaction of the two regions at a distance. Therefore, their results are not necessarily in conflict with ours.…”

“…Martin et al (23) carried out suppressor analysis to obtain information on interactions between specific segments of the largest (B220 or RPB1) and the second-largest (B150 or RPB2) subunits of S. cerevisiae RNA polymerase II. They used two temperature-sensitive mutations, one located near the carboxyl terminus of B220 (in region H) and the other located near the carboxyl terminus of B150 (in region I).…”

Molecular assembly of RNA polymerase II from the fission yeast Schizosaccharomyces pombe: subunit–subunit contact network involving Rpb5

“…Up to now, subunit-subunit contacts within RNA polymerase II have been suggested mainly by mutant studies with the yeast S. cerevisiae and by co-expression studies of subunits from Arabdopsis thaliana. For instance, subunit-subunit interactions have been suggested between RPB1 and RPB2 (Martin et al 1990) and between RPB1 and RPB6 (Archambault et al 1990) of S. cerevisiae RNA polymerase II; and between B36 (RPB3 homologue) and B13.6 (RPB11 homologue) of A. thaliana RNA polymerase II (Ulmasov et al 1996). An understanding of the subunit-subunit network within RNA polymerase II may be useful in making up experimental strategies for the establishment of an in vitro reconstitution system of this multisubunit complex enzyme.…”

Termination-altering amino acid substitutions in the beta' subunit of Escherichia coli RNA polymerase identify regions involved in RNA chain elongation.

“…Conversely two extragenic suppressors of the GD1347 conditional-lethal substitution in interval H of the yeast 13' homolog [ Fig. 5) occur in conserved region I of yeast 13 homolog [rpb2-510(SL1145) and rpb2-513(DNl125); Martin et al 1990]. Thus, the two carboxy-terminal regions of 13 and 13' appear either to interact structurally or to affect the same mechanistic Cold Spring Harbor Laboratory Press on May 12, 2018 -Published by genesdev.cshlp.org Downloaded from step in transcription.…”