Discrimination against deoxyribonucleotide substrates by bacterial RNA polymerase

Nucleic acid polymerases have evolved elaborate mechanisms that prevent incorporation of the non-cognate substrates, which are distinguished by both the base and the sugar moieties. While the mechanisms of substrate selection have been studied in single-subunit DNA and RNA polymerases (DNAPs and RNAPs, respectively), the determinants of substrate binding in the multisubunit RNAPs are not yet known. Molecular modeling of Thermus thermophilus RNAP-substrate NTP complex identified a conserved beta' subunit Asn(737) residue in the active site that could play an essential role in selection of the substrate ribose. We utilized the Escherichia coli RNAP model system to assess this prediction. Functional in vitro analysis demonstrates that the substitutions of the corresponding beta' Asn(458) residue lead to the loss of discrimination between ribo- and deoxyribonucleotide substrates as well as to defects in RNA chain extension. Thus, in contrast to the mechanism utilized by the single-subunit T7 RNAP where substrate selection commences in the inactive pre-insertion site prior to its delivery to the catalytic center, the bacterial RNAPs likely recognize the sugar moiety in the active (insertion) site.     

Distinctive protein signatures provide molecular markers and evidence for the monophyletic nature of the deinococcus-thermus phylum

The Deinococcus-Thermus group of species is currently recognized as a distinct phylum solely on the basis of their branching in 16S rRNA trees. No unique biochemical or molecular characteristics that can distinguish this group from all other bacteria are known at present. In this work, we describe eight conserved indels (viz., inserts or deletions) in seven widely distributed proteins that are distinctive characteristics of the Deinococcus-Thermus phylum but are not found in any other group of bacteria. The identified signatures include a 7-amino-acid (aa) insert in threonyl-tRNA synthetase, 1- and 3-aa inserts in the RNA polymerase beta' subunit, a 5-aa deletion in signal recognition particle (Ffh/SR54), a 2-aa insert in major sigma factor 70 (sigma70), a 2-aa insert in seryl-tRNA synthetase (SerRS), a 1-aa insert in ribosomal protein L1, and a 2-aa insert in UvrA homologs. By using PCR primers for conserved regions, fragments of these genes were amplified from a number of Deinococcus-Thermus species, and all such fragments (except SerRS in Deinococcus proteolyticus) were found to contain the indicated signatures. The presence of these signatures in various species from all three known genera within this phylum, viz., Deinococcus, Thermus, and Meiothermus, provide evidence that they are likely distinctive characteristics of the entire phylum which were introduced in a common ancestor of this group. The signature in SerRS, which is absent in D. proteolyticus, was likely introduced after the branching of this species. Phylogenetic studies as well as the nature of the inserts in some of these proteins (viz., sigma70 and SerRS) also support a sister group relationship between the Thermus and the Meiothermus genera. The identified signatures provide strong evidence for the monophyletic nature of the Deinococcus-Thermus phylum. These molecular markers should prove very useful in the identification of new species related to this group.     

Evolutionary relationships among eubacteria, cyanobacteria, and chloroplasts: evidence from the rpoC1 gene of Anabaena sp. strain PCC 7120.

RNA polymerases of cyanobacteria contain a novel core subunit, gamma, which is absent from the RNA polymerases of other eubacteria. The genes encoding the three largest subunits of RNA polymerase, including gamma, have been isolated from the cyanobacterium Anabaena sp. strain PCC 7120. The genes are linked in the order rpoB, rpoC1, rpoC2 and encode the beta, gamma, and beta' subunits, respectively. These genes are analogous to the rpoBC operon of Escherichia coli, but the functions of rpoC have been split in Anabaena between two genes, rpoC1 and rpoC2. The DNA sequence of the rpoC1 gene was determined and shows that the gamma subunit corresponds to the amino-terminal half of the E. coli beta' subunit. The gamma protein contains several conserved domains found in the largest subunits of all bacterial and eukaryotic RNA polymerases, including a potential zinc finger motif. The spliced rpoC1 gene from spinach chloroplast DNA was expressed in E. coli and shown to encode a protein immunologically related to Anabaena gamma. The similarities in the RNA polymerase gene products and gene organizations between cyanobacteria and chloroplasts support the cyanobacterial origin of chloroplasts and a divergent evolutionary pathway among eubacteria.     

Revealing secrets of the enigmatic omega subunit of bacterial RNA polymerase

The conserved omega (ω) subunit of RNA polymerase (RNAP) is the only nonessential subunit of bacterial RNAP core. The small ω subunit (7 kDa–11.5 kDa) contains three conserved α helices, and helices α2 and α3 contain five fully conserved amino acids of ω. Four conserved amino acids stabilize the correct folding of the ω subunit and one is located in the vicinity of the β′ subunit of RNAP. Otherwise ω shows high variation between bacterial taxa, and although the main interaction partner of ω is always β′, many interactions are taxon‐specific. ω‐less strains show pleiotropic phenotypes, and based on in vivo and in vitro results, a few roles for the ω subunits have been described. Interactions of the ω subunit with the β′ subunit are important for the RNAP core assembly and integrity. In addition, the ω subunit plays a role in promoter selection, as ω‐less RNAP cores recruit fewer primary σ factors and more alternative σ factors than intact RNAP cores in many species. Furthermore, the promoter selection of an ω‐less RNAP holoenzyme bearing the primary σ factor seems to differ from that of an intact RNAP holoenzyme.     

The evolving story of the omega subunit of bacterial RNA polymerase

Omega (omega) is the smallest subunit of bacterial RNA polymerase (RNAP). Although identified early in RNAP research, its function remained ambiguous and shrouded by controversy for a considerable period. It has subsequently been shown that the protein has a structural role in maintenance of the conformation of the largest subunit, beta', and recruitment of beta' to the enzyme assembly. Conservation of this function across all forms of life indicates the importance of its role. Several recent observations have suggested additional functional roles for this protein and have settled some long-standing controversies surrounding it. In this context, revisiting the omega subunit story is especially interesting; here, we review the progress of omega research since its discovery and highlight the importance of these recent observations.     

Normal-mode analysis suggests protein flexibility modulation throughout RNA polymerase's functional cycle

To explore the domain-scale flexibility of bacterial RNA polymerase (RNAP) throughout its functional cycle, block normal-mode analyses (BNM) were performed on several important functional states, including the holoenzyme, the core complex, a model of RNAP bound to primarily duplex DNA, and a model of the ternary elongation complex. The calculations utilized a molecular mechanics (MM) force field with physical interactions; this is made possible by the use of BNM and the implementation of a sparse-matrix diagonalization routine. The use of homology models necessitated the MM force field rather than the simpler elastic network model (ENM). From the MM/BNM, we have systematically and semiquantitatively calculated the atomic fluctuations in the four functional states without bias due to crystal packing or other artifactual forces. We have observed that both alpha subunits and the omega subunit are rigid, in line with their roles as structural motifs that are not mechanistically involved in RNAP's functional cycle. It has been observed that the beta subunit has two highly mobile domains; these are commonly known as the beta1 and beta2 domains. Our calculations suggest that the flexibility of these domains is modulated throughout the functional cycle and that they move entirely independently of each other unless DNA is bound. From an energetic perspective, we have shown the beta2 domain can flex into and out of the cleft, forming interactions with DNA in the TEC as has been previously proposed. Our calculations also confirm that the beta' subunit's likely flexibility into and out of the DNA binding cleft is energetically allowed. These two observations validate that both of the RNAP crab claw's pincers are mobile, as both beta and beta' have substantial flexibility.     

Structural basis of DNA recognition by the alternative sigma-factor, sigma54

The sigma subunit of bacterial RNA polymerase (RNAP) regulates gene expression by directing RNAP to specific promoters. Unlike sigma(70)-type proteins, the alternative sigma factor, sigma(54), requires interaction with an ATPase to open DNA. We present the solution structure of the C-terminal domain of sigma(54) bound to the -24 promoter element, in which the conserved RpoN box motif inserts into the major groove of the DNA. This structure elucidates the basis for sequence specific recognition of the -24 element, orients sigma(54) on the promoter, and suggests how the C-terminal domain of sigma(54) interacts with RNAP.     

Characterization of the subunits of beta-conglycinin

Four subunits of beta-conglycinin were purified from soybean cultivar CX 635-1-1-1, and were designated alpha, alpha', beta, and beta' in accordance with nomenclature proposed by Thanh and Shibasaki [(1977) Biochim. Biophys. Acta 490, 370-384]. Of these subunits, beta' has not previously been reported or characterized. Consistent with the low levels of methionine in these proteins, cyanogen bromide cleavage of alpha', alpha, and beta' subunits produced only a few fragments. The beta subunit contains no methionine and was not cleaved by cyanogen bromide. The NH2-terminal amino acid sequences of the alpha and alpha' subunits are homologous, and each has valine at its amino terminus. The beta subunit has a very different NH2-terminal sequence from those of the alpha and alpha' subunits, and has leucine at its amino terminus. The NH2-terminal sequence of the beta' subunit could not be determined, as it appeared to be blocked to Edman degradation. Although alpha and alpha' subunits have similar NH2-terminal sequences, they differ in the number of methionine residues and so yielded different numbers of cyanogen bromide fragments. Two cyanogen bromide fragments (CB-1 and CB-2) were purified from the alpha subunit. CB-1 originated from the NH2-terminal end of the subunit. The amino acid sequence of CB-2 was identical to that predicted from the nucleotide sequence of cDNA clone pB36. The insert in pB36 encoded 216 amino acids from the COOH-terminal end of the alpha subunit and contained a 138-bp trailer sequence which was followed by a poly-(A) tail. Maps showing the relative positions of methionine residues and carbohydrate moieties in the alpha and alpha' subunits were drawn, based on primary sequence data, and the size and carbohydrate content of the CNBr fragments derived from the subunits.     

Inhibitory effect of phosphate on in vitro development of 2-cell rat embryos is overcome by a factor(s) in oviductal extracts

The promoter recognition site on the sigma70 initiation factor is shielded from interaction with DNA unless sigma70 is bound to the core component of RNA polymerase (RNAP). It is shown that interaction of sigma70 with the isolated beta' subunit of Escherichia coli RNAP is sufficient to induce unshielding of the DNA binding site. Using UV-induced DNA-protein cross-linking we demonstrate that free beta' stimulates specific cross-links between region 2 of the sigma70 polypeptide and a fragment of the non-template promoter strand containing the TATAAT sequence. Thus the sigmabeta' subassembly of RNAP can assume a functionally competent conformation independently of the bulk of the RNAP core.