Isolation, purification, and in vitro characterization of recessive-lethal-mutant RNA polymerases from Escherichia coli

Abstract

The ␤ subunit of prokaryotic RNA polymerase shares significant sequence similarity with its eukaryotic and archaeal counterparts across most of the protein. Nine segments of particularly high similarity have been identified and are termed segments A through I. We have isolated severely defective Escherichia coli RNA polymerase mutants, most of which are unable to support bacterial growth. The majority of the substitutions affect residues in one of the conserved segments of ␤, including invariant residues in segments D (amino acids 548 to 577), E (amino acids 660 to 678), and I (amino acids 1198 to 1296). In addition, recessive-lethal mutations that affect residues highly conserved only among prokaryotes were identified. They include a substitution in the extreme amino terminus of ␤, a region in which no substitutions have previously been identified, and one rpoB mutation that truncates the polypeptide without abolishing minimal polymerase function in vitro. To examine the recessive-lethal alleles in vitro, we devised a novel method to remove nonmutant enzyme from RNA polymerase preparations by affinity tagging the chromosomal rpoB gene. In vitro examination of a subset of purified recessive-lethal RNA polymerases revealed that several substitutions, including all of those altering conserved residues in segment I, severely decrease transcript elongation and increase termination. We discuss the insights these mutants lend to a structure-function analysis of RNA polymerase.Cellular RNA polymerase is a key enzyme in gene expression and regulation. It not only carries out transcription but functions as a central receptor, integrating environmental signals to regulate gene expression. Identification and dissection of structurally and functionally important domains in the enzyme are necessary for an understanding of these processes.The most thoroughly studied cellular RNA polymerase is that of Escherichia coli. Bacterial RNA polymerases are multisubunit enzymes that exist in two forms. The core RNA polymerase (␣ 2 ␤␤Ј) carries out elongation and termination, whereas holoenzyme (␣ 2 ␤␤Ј) carries out initiation (for a review, see reference 3). Both the ␤ and ␤Ј subunits of E. coli RNA polymerase exhibit a high degree of sequence conservation over their entire length when compared with their eukaryotic counterparts (1, 8, 36). In the case of ␤, nine highly conserved segments have been identified, with an average identity of roughly 35% and an average similarity of roughly 70% (36). Given this extensive homology, a structure-function analysis of the E. coli enzyme should provide insight into the organization of all cellular RNA polymerases, as well as into those features of RNA polymerase function that are specific to prokaryotes.Because of the complexity of this essential enzyme, progress in determining its structural features has necessarily been slow.Low-resolution pictures of both E. coli and Saccharomyces cerevisae RNA polymerases have been provided by two-dimensional crystallographic studies (6, 7). These studies ind...

Trans‐dominant mutations in the 3′‐terminal region of the rpoB gene define highly conserved, essential residues in the β subunit of RNA polymerase: the GEME motif

“…A dominant-lethal phenotype was found with RNA polymerase mutants altered in transcriptional propagation that carry certain substitutions in the conserved region surrounding K1065, one of the three C-terminal residues identified by affinity labelling studies (Grachev et al 1987(Grachev et al , 1989Sagitov et al 1993). Recently, a number of recessive-lethal rpoB mutants with increased termination were reported (Tavormina et al 1996). Of the four substitutions in the C-terminal 116 residues of the ␤ subunit described by Tavormina et al (1996), two of the substitutions were in identical residues to those we have identified (H1237Y and E1272K; GEME motif site underlined) and resulted in RNA polymerases with a markedly reduced elongation rate in vitro.…”

“…Recently, a number of recessive-lethal rpoB mutants with increased termination were reported (Tavormina et al 1996). Of the four substitutions in the C-terminal 116 residues of the ␤ subunit described by Tavormina et al (1996), two of the substitutions were in identical residues to those we have identified (H1237Y and E1272K; GEME motif site underlined) and resulted in RNA polymerases with a markedly reduced elongation rate in vitro. Assembly determinants for the ␣ and ␤ 0 subunits are reported to reside in the conserved region encompassing residues 907→1246 and the extreme C-terminus, respectively (Wang et al 1997), and we have identified an assembly patch close to the affinity labelled residue H1237 (Malik et al, manuscript in preparation).…”

Intragenic suppression of trans‐dominant lethal substitutions in the conserved GEME motif of the β subunit of RNA polymerase: evidence for functional cooperativity within the C‐terminus

“…The two valine-substituted enzymes (␤{G1271V} and ␤{M1273V}) give a weak run-off in vitro and also promoter blockade: the enzyme containing ␤{G1271E} is defective in transcriptional run-offs but capable of promoter blockading in vitro (Cromie et al 1999). In a study by Tavormina et al (1996a), the substitution E1272K, one of four recessive lethal missense mutations in the 116-codon 3 0 -terminal region, markedly reduced the elongation rate in vitro. In a very recent mutational analysis of the ␤ subunit, alanine substitution of three of the four GEME residues (i.e.…”

“…The primary substitutions in this region appear to give rise to a form of RNA polymerase that competes efficiently with the wild-type enzyme in a manner deleterious to cell growth, possibly through the production of an RNA polymerase blocked at a stage subsequent to initiation, thereby preventing the wild-type enzyme in a merodiploid cell from functioning (cf. Sagitov et al 1993;Tavormina et al 1996a;Tavormina et al 1996b). Given the nature and proximity of the two substitutions, EEME and GEMV, in this universally conserved motif (Fig.…”

Structural Perspective on Mutations Affecting the Function of Multisubunit RNA Polymerases

“…We speculate that the replacement for a serine could lead to a promoter opening defect and therefore to a low polymerization rate. Similarly, replacements Q393P (81,82,202), conferring rifampin resistance, and G446D (174,175), isolated based on its increased termination phenotype, both surround fork loop 2 and possibly destabilize the structure of this element resulting in the inability to maintain the transcription bubble open.…”