Chlamydia trachomatis RNA polymerase alpha subunit: sequence and structural analysis

Abstract

We describe the cloning and sequence analysis of the region surrounding the gene for the ␣ subunit of RNA polymerase from Chlamydia trachomatis. This region contains genes for proteins in the order SecY, S13, S11, ␣, and L17, which are equivalent to Escherichia coli and Bacillus subtilis r proteins. The incorporation of chlamydial ␣ subunit protein into the E. coli RNA polymerase holoenzyme rather than its truncated variant lacking the amino terminus suggests the existence of structural conservation among ␣ subunits from distantly related genera.The obligate intracellular bacterium Chlamydia trachomatis is a major human pathogen responsible for a wide range of infections (37). It exhibits a unique developmental cycle alternating between noninfectious intracellular, metabolically active reticulate bodies and infectious extracellular, inert elementary bodies (EB). The process of chlamydial differentiation and development is complex and involves temporally regulated genes. Both early-and late-development-specific genes have been identified; however, the mechanism underlying stagespecific gene expression remains unelucidated (3,11,30,43,47).A better understanding of chlamydial gene regulation will require a more detailed characterization of its RNA polymerase (RNAP). Studies of RNAP subunits have contributed to our understanding of the mechanism of gene regulation in Escherichia coli (12). The bacterial DNA-dependent RNAPs are complex enzymes comprising four different subunits with the subunit structure ␣ 2 ␤␤Ј that perform various functions leading to regulated transcription (12,15,16). Assembly of the core RNAP takes place in a sequential manner in the order ␣ 3 ␣ 2 3 ␣ 2 ␤ 3 ␣ 2 ␤␤Ј (15). The core enzyme is active in RNA polymerization, while the binding of confers the promoter specificity to the holoenzyme. The purification of RNAP from Chlamydia spp. is a difficult process because of the limitations imposed by very little starting material. Recently Mathews et al. (25) studied the in vitro transcription of chlamydial genes by using a crude preparation of native RNAP. Recent work has focused on the cloning and sequencing of various RNAP subunits with the aim of developing an in vitro transcription system to study surrogate chlamydial genetics.Two groups have independently reported the cloning and sequencing of 66 , ␤, and ␤Ј homologs from Chlamydia spp. (6,7,21). While this work was in progress, Tan et al. (42) reported the cloning and sequence analysis of the ␣ subunit from the C. trachomatis mouse pneumonitis biovar (MoPn). However, these studies have been found to be in error, and the sequence described applies to Mycoplasma spp. rather than Chlamydia spp. (see the author's correction in this issue).Cloning and genomic organization of C. trachomatis ␣ operon genes. The purpose of this investigation was to clone and characterize the ␣ subunit of the RNAP holoenzyme with the aim of developing an in vitro transcription system in which purified and reconstituted RNAP are used. We have previously isolated the ...

DNA-binding determinants of the alpha subunit of RNA polymerase: novel DNA-binding domain architecture.

“…2). Six of these seven amino acid residues are invariant in all known bacterial a-subunit sequences (Gebhardt et al 1993;Gu et al 1995; GenBank accession nos. U32762 and L42023).…”

Identification of sequences necessary for transcription in vitro from the Chlamydia trachomatis rRNA P1 promoter

“…Explanations for this sequence diversity include the possibilities that (i) some of the presumptive chlamydial transcription initiation sites are instead processing sites, (ii) the putative promoters belong to more than one class of promoters, and (iii) there is latitude in the promoter specificity of C. trachomatis A RNA polymerase (RNAP). The subunits of C. trachomatis RNAP have been cloned and sequenced (9,10,18,21).A , a C. trachomatis 70 homolog (9, 21), is the only sigma factor identified in chlamydiae to date.A and 70 share striking amino acid sequence conservation in subregions 2.4 and 4.2 (9, 21) of 70 , including the specific amino acid residues which have been shown in E. coli to be involved in promoter recognition at the Ϫ10 and Ϫ35 positions (reviewed in reference 5). This conservation of amino acid residues involved in promoter recognition contrasts with the observed variability in the DNA sequences of the putative C. trachomatis promoters.…”

“…Explanations for this sequence diversity include the possibilities that (i) some of the presumptive chlamydial transcription initiation sites are instead processing sites, (ii) the putative promoters belong to more than one class of promoters, and (iii) there is latitude in the promoter specificity of C. trachomatis A RNA polymerase (RNAP). The subunits of C. trachomatis RNAP have been cloned and sequenced (9,10,18,21).…”

The RNA Polymerase α Subunit from Sinorhizobium meliloti Can Assemble with RNA Polymerase Subunits from Escherichia coli and Function in Basal and Activated Transcription both In Vivo and In Vitro

“…However, rpsD is not part of this operon in S. meliloti, B. subtilis, Mesorhizobium loti, or Rickettsia prowazekii (3,11,37), indicating that S4 may not regulate this operon in these species. The second gene in this region in S. meliloti, adk, which encodes adenylate kinase, is not found in the rpoA region in most bacterial species for which this region has been sequenced (30,31,49). Based both on comparisons with the DNA sequences for E. coli and B. subtilis and on the lack of a predicted transcriptional terminator between any of the genes we sequenced, we hypothesize that at least some S. meliloti rpoA transcription must be initiated from a promoter upstream of secY, which is part of the spc operon (16,60) and which is probably cotranscribed with ribosomal protein genes.…”