Functional complementation between mutations at two distant positions in Escherichia coli RNA polymerase as revealed by second-site suppression

Abstract

Subunit-subunit interactions are critical for the assembly of the core of Escherichia coli RNA polymerase. The mutant a-subunit C131A is unable to complement the temperature-sensitive a-R45C mutant strain, which is defective for binding of the b-subunit. In vitro reconstitution experiments, however, indicate that the a-C131A variant is able to form the intermediate a 2 b, but is defective in contacting the b¢-subunit. We used this a-C131A mutant to isolate a suppressor mutation in the b¢-subunit. Genetic and biochemical characterization of the b¢ suppressor indicates the allelespeci®c nature of its eect. Sequence analysis of the suppressor revealed a single substitution of Gly at position 333, an evolutionarily conserved position in the conserved region C of the b¢-subunit, by Asp. However, the crystal structure of the bacterial RNA polymerase indicates that the primary mutation (a-C131A) and its suppressor lie far apart. Thus, we propose that longrange interactions, as in this case, may play an important role in the functional assembly of E. coli RNA polymerase.

Functional Interplay between the Jaw Domain of Bacterial RNA Polymerase and Allele-specific Residues in the Product RNA-binding Pocket

“…In vitro elongation rate measurements. Transcription reactions (Materials and Methods) using wild-type or mutant RNAPs indicated above the panels were performed using initially halted TECs (0 lanes) and sampled at 5,10,15,20,25,30,45,60,90, 120, and 240 s (arrows). The average time of appearance of the run-off transcript (668 nt) as used to estimate elongation rate (average of three experiments of which the data shown are representative).…”

“…21,22 Because rif R mutants can be readily generated and then screened for a particular phenotype, they have proven useful in characterizing the ways in which RNAP activity can be altered and in finding suppressors of other phenotypes. 13,[23][24][25][26] Genetic suppression has proven to be a powerful tool to dissect RNAP function [27][28][29][30] and is wellsuited to investigate jaw-domain function, which likely involves long-range interactions within RNAP in addition to primary contacts. Crystal structures of RNAP and its transcription complexes reveal interaction with the jaw of a mobile b 0 loop called the trigger loop whose effects on the active site may be altered when the jaw is deleted.…”

Structure–function–rescue: the diverse nature of common p53 cancer mutants

“…Besides the importance for understanding evolutionary processes that minimize loss of fitness, the analysis of secondsite suppressor mutations is also a powerful tool for studying functional interactions within and among proteins (Hartman and Roth, 1973;Poteete et al, 1991;Wray et al, 1999;Sujatha et al, 2001). In the case of p53, studies on second-site suppressor mutations are particularly interesting, because they provide important clues as to whether activity can be restored to common cancer mutants, which has far-reaching consequences for the development of therapeutic anticancer strategies.…”

Stabilizing interactions in the dimer interface of α‐subunit in Escherichia coli RNA polymerase: A graph spectral and point mutation study

“…The graph spectral analysis here was performed with the crystal structure coordinates obtained from the α 2 dimer of E. coli and from the Taq RNAP. The consistency in results from either of the data sets indicates that β or β′ assembly over α‐dimer may not influence the core structure at the dimer interface (Kimura et al 1994; Sujatha and Chatterji 1999; Sujatha et al 2000).…”