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.     

Structure and function of lineage-specific sequence insertions in the bacterial RNA polymerase beta' subunit

The large beta and beta' subunits of the bacterial core RNA polymerase (RNAP) are highly conserved throughout evolution. Nevertheless, large sequence insertions in beta and beta' characterize specific evolutionary lineages of bacteria. The Thermus aquaticus RNAP beta' subunit contains a 283 residue insert between conserved regions A and B that is found in only four bacterial species. The Escherichia coli RNAP beta' subunit contains a 188 residue insert in the middle of conserved region G that is found in a wide range of bacterial species. Here, we present structural studies of these two beta' insertions. We show that the inserts comprise repeats of a previously characterized fold, the sandwich-barrel hybrid motif (as predicted from previous sequence analysis) and that the inserts serve significant roles in facilitating protein/protein and/or protein/nucleic acid interactions.     

Inter-subunit recognition and manifestation of segmental mobility in Escherichia coli RNA polymerase: a case study with omega-beta' interaction

Omega (omega), consisting of 91 amino acids, is the smallest of all the Escherichia coli RNA polymerase subunits and is organized into an N-terminal domain of 53 amino acids followed by an unstructured tail in the C-terminal region. Our earlier experiments have shown a chaperone-like function of omega in which it helps to maintain beta' in a correct conformation and recruit it to the alpha(2)beta subassembly to form a functional core enzyme (alpha(2)betabeta'omega). The X-ray structure analysis of Thermus aquaticus core RNA polymerase suggests that two regions of omega latch onto the N-terminal and C-terminal ends of the beta'-subunit. In the present study we have monitored the conformational changes in beta' as the denatured protein is refolded in the presence and absence of omega using tryptophan fluorescence emission of beta' as well as acrylamide quenching of Trp fluorescence. Results indicate that the presence of stoichiometric amounts of omega is helpful in beta' refolding. We have also monitored the behavior of the C-terminal tail of omega by engineering three cysteine residues at three different sites in omega and subsequently labeling them with a sulphydryl-specific fluorescent probe. Fluorescence anisotropy measurements of the labeled protein indicate that the C-terminal domain of omega is mobile in the free protein and gets restrained in the presence of beta'. Calculations on side-chain interactions show that out of the three mutated positions, two have near neighbourhood interactions only with side-chains in the beta' subunit whereas the end of the C-terminal of omega, although it is restrained in the presence of beta', has no interacting partner within a 4-A radius.     

Escherichia coli RNA polymerase subunit omega and its N-terminal domain bind full-length beta' to facilitate incorporation into the alpha2beta subassembly.

The omega subunit of Escherichia coli RNA polymerase, consisting of 90 amino acids, is present in stoichiometric amounts per molecule of core RNA polymerase (alpha2betabeta'). The presence of omega is necessary to restore denatured RNA polymerase in vitro to its fully functional form, and, in an omega-less strain of E. coli, GroEL appears to substitute for omega in the maturation of RNA polymerase. The X-ray structure of Thermus aquaticus core RNA polymerase suggests that two regions of omega latch on to beta' at its N-terminus and C-terminus. We show here that omega binds only the intact beta' subunit and not the beta' N-terminal domain or beta' C-terminal domain, implying that omega binding requires both these regions of beta'. We further show that omega can prevent the aggregation of beta' during its renaturation in vitro and that a V8-protease-resistant 52-amino-acid-long N-terminal domain of omega is sufficient for binding and renaturation of beta'. CD and functional assays show that this N-terminal fragment retains the structure of native omega and is able to enhance the reconstitution of core RNA polymerase. Reconstitution of core RNA polymerase from its individual subunits proceeds according to the steps alpha + alpha --> alpha2 + beta --> alpha2beta + beta' --> alpha2betabeta'. It is shown here that omega participates during the last stage of enzyme assembly when beta' associates with the alpha2beta subassembly.     

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.     

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.     

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.