Irina Bass

Unified two-metal mechanism of RNA synthesis and degradation by RNA polymerase.

In DNA‐dependent RNA polymerases, reactions of RNA synthesis and degradation are performed by the same active center (in contrast to DNA polymerases in which they are separate). We propose a unified catalytic mechanism for multisubunit RNA polymerases based on the analysis of its 3′–5′ exonuclease reaction in the context of crystal structure. The active center involves a symmetrical pair of Mg2+ ions that switch roles in synthesis and degradation. One ion is retained permanently and the other is recruited ad hoc for each act of catalysis. The weakly bound Mg2+ is stabilized in the active center in different modes depending on the type of reaction: during synthesis by the β,γ‐phosphates of the incoming substrate; and during hydrolysis by the phosphates of a non‐base‐paired nucleoside triphosphate. The latter mode defines a transient, non‐specific nucleoside triphosphate‐binding site adjacent to the active center, which may serve as a gateway for polymerization of substrates.

Four genes, two ends, and a res region are involved in transposition of Tn5053: a paradigm for a novel family of transposons carrying either a mer operon or an integron

The complete nucleotide sequence of an 8447 bp‐long mercury‐resistance transposon (Tn5053) has been determined. Tn5053 is composed of two modules: (i) the mercury‐resistance module and (ii) the transposition module. The mercury‐resistance module carries a mer operon, merRTPFAD, and appears to be a single‐ended relic of a transposon closely related to the classical mercury‐resistance transposons Tn21 and Tn501. The transposition module of Tn5053 is bounded by 25 bp terminal inverted repeats and contains four genes involved in transposition, i.e. tniA, tniB, tniQ, and tniR. Transposition of Tn5053 occurs via cointegrate formation mediated by the products of the tniABQ genes, followed by site‐specific cointegrate resolution. This is catalysed by the product of the tniR gene at the res region, which is located upstream of tniR. The same pathway of transposition is used by Tn402 (Tn5090) which carries the integron of R751. Transposition genes of Tn5053 and Tn402 are interchangeable. Sequence analysis suggests that Tn5053 and Tn402 are representatives of a new family of transposable elements, which fall into a recently recognized superfamily of transposons including retroviruses, insertion sequences of the IS3 family, and transposons Tn552 and Tn7. We suggest that the tni genes were involved in the dissemination of integrons.

RNA polymerase rifampicin resistance mutations in Escherichia coli: Sequence changes and dominance

SummaryFive recombinant plasmids, pBK2646, pBK611, pRC3, pRC4 and pRC5, carrying rpoB rifampicin-resistant RNA-polymerase genes were obtained.The sequence analysis of these plasmids revealed certain structural changes in the rpoB gene which specify corresponding alterations in the β-subunit of RNA polymerase. Some functional properties of the corresponding mutant strains and their RNA polymerases have been investigated.

Structural Modules of the Large Subunits of RNA Polymerase INTRODUCING ARCHAEBACTERIAL AND CHLOROPLAST SPLIT SITES IN THE β AND β′ SUBUNITS OF ESCHERICHIA COLI RNA POLYMERASE

The β and β′ subunits of Escherichia coli DNA-dependent RNA polymerase are highly conserved throughout eubacterial and eukaryotic kingdoms. However, in some archaebacteria and chloroplasts, the corresponding sequences are “split” into smaller polypeptides that are encoded by separate genes. To test if such split sites can be accommodated into E. coli RNA polymerase, subunit fragments encoded by the segments of E. coli rpoB and rpoC genes corresponding to archaebacterial and chloroplast split subunits were individually overexpressed. The purified fragments, when mixed in vitro with complementing intact RNA polymerase subunits, yielded an active enzyme capable of catalyzing the phosphodiester bond formation. Thus, the large subunits of eubacteria and eukaryotes are composed of independent structural modules corresponding to the smaller subunits of archaebacteria and chloroplasts.

Horizontal spread of mer operons among gram-positive bacteria in natural environments.

Horizontal dissemination of the genes responsible for resistance to toxic pollutants may play a key role in the adaptation of bacterial populations to environmental contaminants. However, the frequency and extent of gene dissemination in natural environments is not known. A natural horizontal spread of two distinct mercury resistance (mer) operon variants, which occurred amongst diverse Bacillus and related species over wide geographical areas, is reported. One mer variant encodes a mercuric reductase with a single N-terminal domain, whilst the other encodes a reductase with a duplicated N-terminal domain. The strains containing the former mer operon types are sensitive to organomercurials, and are most common in the terrestrial mercury-resistant Bacillus populations studied in this work. The strains containing the latter operon types are resistant to organomercurials, and dominate in a Minamata Bay mercury-resistant Bacillus population, previously described in the literature. At least three distinct transposons (related to a class II vancomycin-resistance transposon, Tn1546, from a clinical Enterococcus strain) and conjugative plasmids are implicated as mediators of the spread of these mer operons.

Tn5041: a chimeric mercury resistance transposon closely related to the toluene degradative transposon Tn4651

This paper reports the discovery and characterization of Tn5041, a novel-type transposon vehicle for dissemination of mercury resistance in natural bacterial populations. Tn5041 (14876 bp), identified in a Pseudomonas strain from a mercury mine, is a Tn3 family mercury resistance transposon far outside the Tn21 subgroup. As in other Tn3 family transposons, Tn5041 duplicates 5 bp of the target sequence following insertion. Tn5041 apparently acquired its mer operon as a single-ended relic of a transposon belonging to the classical mercury resistance transposons of the Tn21 subgroup. The putative transposase and the 47 bp terminal inverted repeats of Tn5041 are closely related to those of the toluene degradative transposon Tn4651 and fall into a distinct subgroup on the fringe of the Tn3 family. The amino acid sequence of the putative resolvase of Tn5041 resembles site-specific recombinases of the integrase family. Besides the mer operon and putative transposition genes, Tn5041 contains a 4 kb region that accommodates a number of apparently defective genes and mobile elements.

Region 1.2 of the RNA polymerase σ subunit controls recognition of the −10 promoter element

Recognition of the −10 promoter consensus element by region 2 of the bacterial RNA polymerase σ subunit is a key step in transcription initiation. σ also functions as an elongation factor, inducing transcription pausing by interacting with transcribed DNA non‐template strand sequences that are similar to the −10 element sequence. Here, we show that the region 1.2 of Escherichia coli σ70, whose function was heretofore unknown, is strictly required for efficient recognition of the non‐template strand of −10‐like pause‐inducing DNA sequence by σ region 2, and for σ‐dependent promoter‐proximal pausing. Recognition of the fork‐junction promoter DNA by RNA polymerase holoenzyme also requires σ region 1.2 and thus resembles the pause‐inducing sequence recognition. Our results, together with available structural data, support a model where σ region 1.2 acts as a core RNA polymerase‐dependent allosteric switch that modulates non‐template DNA strand recognition by σ region 2 during transcription initiation and elongation.

Distinct functions of the RNA polymerase σ subunit region 3.2 in RNA priming and promoter escape.

The σ subunit of bacterial RNA polymerase (RNAP) has been implicated in all steps of transcription initiation, including promoter recognition and opening, priming of RNA synthesis, abortive initiation and promoter escape. The post-promoter-recognition σ functions were proposed to depend on its conserved region σ3.2 that directly contacts promoter DNA immediately upstream of the RNAP active centre and occupies the RNA exit path. Analysis of the transcription effects of substitutions and deletions in this region in Escherichia coli σ70 subunit, performed in this work, suggests that (i) individual residues in the σ3.2 finger collectively contribute to RNA priming by RNAP, likely by the positioning of the template DNA strand in the active centre, but are not critical to promoter escape; (ii) the physical presence of σ3.2 in the RNA exit channel is important for promoter escape; (iii) σ3.2 promotes σ dissociation during initiation and suppresses σ-dependent promoter-proximal pausing; (iv) σ3.2 contributes to allosteric inhibition of the initiating NTP binding by rifamycins. Thus, region σ3.2 performs distinct functions in transcription initiation and its inhibition by antibiotics. The B-reader element of eukaryotic factor TFIIB likely plays similar roles in RNAPII transcription, revealing common principles in transcription initiation in various domains of life.

Class II broad-spectrum mercury resistance transposons in Gram-positive bacteria from natural environments

We have studied the mechanisms of the horizontal dissemination of a broad-spectrum mercury resistance determinant among Bacillus and related species. This mer determinant was first described in Bacillus cereus RC607 from Boston Harbor, USA, and was then found in various Bacillus and related species in Japan, Russia and England. We have shown that the mer determinant can either be located at the chromosome, or on a plasmid in the Bacillus species, and is carried by class II mercury resistance transposons: Tn5084 from B. cereus RC607 and B. cereus VKM684 (ATCC10702) and Tn5085 from Exiguobacterium sp. TC38-2b. Tn5085 is identical in nucleotide sequence to TnMERI1, the only other known mer transposon from Bacillus species, but it does not contain an intron like TnMERI1. Tn5085 is functionally active in Escherichia coli. Tn5083, which we have isolated from B. megaterium MK64-1, contains an RC607-like mer determinant, that has lost some mercury resistance genes and possesses a merA gene which is a novel sequence variant that has not been previously described. Tn5083 and Tn5084 are recombinants, and are comprised of fragments from several transposons including Tn5085, and a relative of a putative transposon from B. firmus (which contains similar genes to the cadmium resistance operon of Staphylococcus aureus), as well as others. The sequence data showed evidence for recombination both between transposition genes and between mer determinants.

Cold Sensitivity of Thermophilic and Mesophilic RNA Polymerases

RNA polymerase from mesophilic Deinococcus radiodurans displays the same cold sensitivity of promoter opening as RNA polymerase from the closely related thermophilic Thermus aquaticus. This suggests that, contrary to the accepted view, cold sensitivity of promoter opening by thermophilic RNA polymerases may not be a consequence of their thermostability.