To identify regions of the largest subunit of RNA polymerase that are potentially involved in transcript elongation and termination, we have characterized amino acid substitutions in the 13' subunit of Escherichia coli RNA polymerase that alter expression of reporter genes preceded by terminators in vivo. Termination-altering substitutions occurred in discrete segments of [3', designated 2, 3a, 3b, 4a, 4b, 4c, and 5, many of which are highly conserved in eukaryotic homologs of 13'. Region 2 substitutions (residues 311-386) are tightly clustered around a short sequence that is similar to a portion of the DNA-binding cleft in E. coli DNA polymerase I. Region 3b (residues 718-798) corresponds to the segment of the largest subunit of RNA polymerase II in which amanitin-resistance substitutions occur. Region 4a substitutions (residues 933-936) occur in a segment thought to contact the transcript 3' end. Region 5 substitutions (residues 1308-1356) are tightly clustered in conserved region H near the carboxyl terminus of [3'. A representative set of mutant RNA polymerases were purified and revealed unexpected variation in percent termination at six different p-independent terminators. Based on the location and properties of these substitutions, we suggest a hypothesis for the relationship of subunits in the transcription complex.[Key Words: rpoC gene; [3' subunit; RNA polymerase; transcriptional termination] Received July 26, 1994; revised version accepted October 13, 1994.Escherichia coli RNA polymerase contains a core of four subunits: 6', 155 kD; [3, 150 kD; and two c~, 43 kD each. These subunits form a scaffold and catalytic center for synthesis of RNA on a double-stranded DNA template that appear to be conserved from bacteria to humans. The two largest subunits, 6' and B in E. coli, display significant sequence similarity to homologous subunits found in all multisubunit RNA polymerases (Allison et al. 1985; Jokerst et al. 1989; Young 1991 and references therein): Each contains eight to nine conserved, colinear segments, although no sequence conservation is evident between f3' and B. However, we currently lack a clear picture of how these conserved primary sequence motifs are positioned in the three-dimensional structure of RNA polymerase.In the transcription elongation complex (for review, see Das 1993;Chamberlin 1994;Chan and Landick 1994), RNA polymerase contacts the DNA over a 25-to ~Present address:

Interaction of the poly(A) binding protein, Pab1p, with mRNA plays an important role in gene expression. This work describes an analysis of pab1 mutants in Saccharomyces cerevisiae. Yeast pab1 mutants were found to be sensitive to elevated concentrations of copper (Cu) and 3-aminotriazole (3-AT) in the growth medium. This phenotype arises because these pab1 mutants underaccumulate mRNA, including the CUP1 and HIS3 mRNAs, the products of which are required for Cu and 3-AT resistance, respectively. To determine the cause of the mRNA underaccumulation, mRNA turnover and production were examined in the pab1-53 mutant. It was found that although the pattern of mRNA decay was altered, and substantial decapping of polyadenylated mRNA could be detected, mRNA was not destabilized in this strain. It was also found that the pab1 mutant was impaired in the production of mRNA. These data show that the decreased level of mRNA in the pab1-53 mutant arises from poor production, and they suggest that yeast Pab1p is involved in an aspect of nuclear mRNA metabolism. They also indicate that deadenylation can be uncoupled from decapping without significant changes in an mRNA's stability, and that this uncoupling can be tolerated by yeast.

Interaction of the poly(A) binding protein, Pab1p, with mRNA plays an important role in gene expression. This work describes an analysis of pab1 mutants in Saccharomyces cerevisiae. Yeast pab1 mutants were found to be sensitive to elevated concentrations of copper (Cu) and 3‐aminotriazole (3‐AT) in the growth medium. This phenotype arises because these pab1 mutants underaccumulate mRNA, including the CUP1 and HIS3 mRNAs, the products of which are required for Cu and 3‐AT resistance, respectively. To determine the cause of the mRNA underaccumulation, mRNA turnover and production were examined in the pab1‐53 mutant. It was found that although the pattern of mRNA decay was altered, and substantial decapping of polyadenylated mRNA could be detected, mRNA was not destabilized in this strain. It was also found that the pab1 mutant was impaired in the production of mRNA. These data show that the decreased level of mRNA in the pab1‐53 mutant arises from poor production, and they suggest that yeast Pab1p is involved in an aspect of nuclear mRNA metabolism. They also indicate that deadenylation can be uncoupled from decapping without significant changes in an mRNA's stability, and that this uncoupling can be tolerated by yeast. Copyright © 1999 John Wiley & Sons, Ltd.