Assembly of functional Escherichia coli RNA polymerase containing beta subunit fragments.

Severinov, Konstantin; Mustaev, Arkady; Severinova, Elena; Bass, I. A.; Kashlev, Mikhail; Landick, Robert; Nikiforov, Vadim; Goldfarb, Alex; Darst, Seth A.

doi:10.1073/pnas.92.10.4591

Cited by 41 publications

(46 citation statements)

References 55 publications

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“…To determine where the pause hairpin contacts ␤, we first tested crosslinking in transcription complexes in which ␤ was split into two functional pieces (37). One type contained fragments 1-235 and 235-1,342 (S1, Fig.…”

Section: Resultsmentioning

confidence: 99%

Preferential interaction of the his pause RNA hairpin with RNA polymerase β subunit residues 904–950 correlates with strong transcriptional pausing

Wang¹,

Severinov²,

Landick³

1997

Proc. Natl. Acad. Sci. U.S.A.

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RNA secondary structures (hairpins) that form as the nascent RNA emerges from RNA polymerase are important components of many signals that regulate transcription, including some pause sites, all -independent terminators, and some antiterminators. At the his leader pause site, a 5-bp-stem, 8-nt-loop pause RNA hairpin forms 11 nt from the RNA 3 end and stabilizes a transcription complex conformation slow to react with NTP substrate. This stabilization appears to depend at least in part on an interaction with RNA polymerase. We tested for RNA hairpin interaction with the paused polymerase by crosslinking 5-iodoUMP positioned specifically in the hairpin loop. In the paused conformation, strong and unusual crosslinking of the pause hairpin to ␤904-950 replaced crosslinking to ␤ and to other parts of ␤ that occurred in nonpaused complexes prior to hairpin formation. These changes in nascent RNA interactions may inhibit reactive alignment of the RNA 3 end in the paused complex and be related to events at -independent terminators.The his leader pause hairpin is a well characterized nascent RNA structure that modulates RNA chain elongation (1-4). It forms concomitantly with a paused conformation of the transcription complex midway through the his biosynthetic operon leader region, where it increases the lifetime of the paused complex at least 6-fold. The pause allows time for a ribosome to initiate synthesis of the leader peptide and release the paused polymerase (probably by disrupting the pause hairpin), thereby synchronizing ribosome and polymerase movement during transcriptional attenuation (5). This and other pause sites are regulatory timing mechanisms that both prevent transcription beyond a region where regulatory input is effective and put polymerase in the proper conformation to interact with regulatory molecules.Similar hairpins are involved in some, but not all, pause signals; are required at -independent terminators (6-8), where they trigger dissociation of the transcription complex; and alone (e.g., HK022 put; ref. 9) or in association with proteins (e.g., N-nut complex; ref. 8) can function as antiterminators, which modify the transcription complex to block recognition of both pause sites and terminators. Nascent RNA hairpins also are components of some eukaryotic regulatory mechanisms (e.g., HIV TAR RNA; ref. 10) and can trigger pausing or termination by RNA polymerase II in vitro (11). Although RNA hairpin-RNA polymerase interactions have long been suggested to play roles in pausing (12), termination (13-15), and antitermination (16), no direct evidence for these interactions or their effects exists.The his pause RNA hairpin offers an excellent paradigm to study these interactions. It is one of four components of the multipartite his pause signal that also depends on a 3Ј-proximal region of RNA or DNA, alignment of the 3Ј-terminal nucleotide and the incoming NTP, and duplex DNA that contacts polymerase downstream from the active site (1, 2). Both the his pause signal and -independent terminators ...

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Section: Resultsmentioning

confidence: 99%

Preferential interaction of the his pause RNA hairpin with RNA polymerase β subunit residues 904–950 correlates with strong transcriptional pausing

Wang¹,

Severinov²,

Landick³

1997

Proc. Natl. Acad. Sci. U.S.A.

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“…DR1 and DR2 of the ␤ subunit are poorly conserved or even missing in ␤ homologs of other organisms, and E. coli RNAP can tolerate large deletions (more than 200 aa in some cases) in these regions without loss of function in vitro (11,12). These properties suggest that the DRs must be structurally autonomous to accommodate such large deletions or insertions without disturbing the central functions of the enzyme.…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, DR1 is targeted by the bacteriophage T4-Alc protein, which selectively induces premature termination of E. coli RNAP transcription on E. coli DNA during infection (12), indicating that regulatory factors can impact transcription through the DRs. Thus, we are left with the interesting picture of the RNAP molecule as a central core structure, contained within an approximately 40-Å radius sphere centered at the active site Mg 2ϩ ion, that is highly conserved in sequence and nearly identical in structure among all organisms (7,9,24,25).…”

Section: Discussionmentioning

confidence: 99%

“…Nevertheless, E. coli ␤Ј lacks a 283-residue domain present in Taq ␤Ј between conserved regions A and B (Taq ␤Ј nonconserved domain 1, or Taq ␤ЈNCD1), and contains a 188-residue domain immediately C terminal of conserved region G (E. coli ␤ЈNCD2) that is missing in Taq. In ␤, the E. coli subunit harbors two large domains missing in the Taq subunit, a 115-residue domain inserted between conserved regions B and C, and a 99-residue domain between conserved regions G and H. Because large deletions in these two regions of E. coli ␤ do not affect RNAP assembly and basic function in vitro, they have been termed dispensable regions I (DR1) and 2 (DR2), respectively (11,12). DR2 of the E. coli ␤ subunit has been localized on the low-resolution structure of core RNAP (13).…”

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confidence: 99%

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Conformational flexibility of bacterial RNA polymerase

Darst

Opalka

Chacón

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

109

113

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The structure of Escherichia coli core RNA polymerase (RNAP) was determined by cryo-electron microscopy and image processing of helical crystals to a nominal resolution of 15 Å. Because of the high sequence conservation between the core RNAP subunits, we were able to interpret the E. coli structure in relation to the highresolution x-ray structure of Thermus aquaticus core RNAP. A very large conformational change of the T. aquaticus RNAP x-ray structure, corresponding to opening of the main DNA͞RNA channel by nearly 25 Å, was required to fit the E. coli map. This finding reveals, at least partially, the range of conformational flexibility of the RNAP, which is likely to have functional implications for the initiation of transcription, where the DNA template must be loaded into the channel.

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“…The assembly pathway should be more complex in organisms with split ␤, ␤Ј homologues, but this has not been investigated. Our own work with E. coli defined four separable domains in ␤ and three in ␤Ј (16,17), that correspond to natural fragmentation sites in some of the archaea and cyanobacteria. The ability to generate functional E. coli RNAP using subunit domains greatly aided the study of RNAP assembly and biochemical functions of assembly intermediates.…”

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confidence: 99%

Inter- and Intrasubunit Interactions during the Formation of RNA Polymerase Assembly Intermediate

Naryshkina¹,

Rogulja²,

Golub³

et al. 2000

Journal of Biological Chemistry

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We used yeast two-hybrid and in vitro co-immobilization assays to study the interaction between the Escherichia coli RNA polymerase (RNAP) ␣ and ␤ subunits during the formation of ␣ 2 ␤, a physiological RNAP assembly intermediate. We show that a 430-amino acidlong fragment containing ␤ conserved segments F, G, H, and a short part of segment I forms a minimal domain capable of specific interaction with ␣. The ␣-interacting domain is held together by protein-protein interactions between ␤ segments F and I. Residues in catalytically important ␤ segments H and I directly participate in ␣ binding; substitutions of strictly conserved segment H Asp 1084 and segment I Gly 1215 abolish ␣ 2 ␤ formation in vitro and are lethal in vivo. The importance of these ␤ amino acids in ␣ binding is fully supported by the structural model of the Thermus aquaticus RNAP core enzyme. We also demonstrate that determinants of RNAP assembly are conserved, and that a homologue of ␤ Asp 1084 in A135, the ␤-like subunit of yeast RNAP I, is responsible for interaction with AC40, the largest ␣-like subunit. However, the A135-AC40 interaction is weak compared with the E. coli ␣-␤ interaction, and A135 mutation that abolishes the interaction is phenotypically silent. The results suggest that in eukaryotes additional RNAP subunits orchestrate the enzyme assembly by stabilizing weak, but specific interactions of core subunits.Cellular RNA polymerases (RNAPs) 1 are large, multisubunit enzymes. A typical prokaryotic RNAP core contains 5 polypeptides with a total molecular mass of ϳ400 kDa. Core RNAP from eukaryotes and archaea contain 10 -14 subunits with a total molecular mass in excess of 500 kDa. Sequence alignments of RNAP subunits reveal extensive similarities; each of the two largest RNAP subunits, which are the most evolutionarily conserved, contains 8 -9 colinear segments with many invariant amino acids (1, 2). RNAPs from different sources are also homologous structurally; low resolution (16 -35 Å) threedimensional models of Escherichia coli, and RNAP II and RNAP I from yeast obtained by means of electron crystallography reveal significant similarities (3-5).Evolutionarily conserved subunit segments probably form distinct functional domains common to all RNAPs. Genetic data support this notion; mutational changes of conserved residues selectively destroy distinct partial functions of the enzyme (e.g. the transition from abortive initiation to productive elongation (Ref. 6), or phosphodiester bond synthesis (Ref. 7)), but leave other functions unperturbed. However, isolated subunits themselves do not possess any of the partial functions of the whole enzyme (8). Therefore, RNAP functional sites are either formed allosterically upon the enzyme assembly, or are located at subunit interfaces. Thus, understanding inter-and intrasubunit interactions should provide insights in RNAP mechanism and regulation.E. coli RNAP assembles in vivo and in vitro according to the following scheme: ␣-␣ 2 -␣ 2 ␤-␣ 2 ␤␤Ј (9). The ␣ 2 ␤ assembly intermediate appears...

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Assembly of functional Escherichia coli RNA polymerase containing beta subunit fragments.

Cited by 41 publications

References 55 publications

Preferential interaction of the his pause RNA hairpin with RNA polymerase β subunit residues 904–950 correlates with strong transcriptional pausing

Preferential interaction of the his pause RNA hairpin with RNA polymerase β subunit residues 904–950 correlates with strong transcriptional pausing

Conformational flexibility of bacterial RNA polymerase

Inter- and Intrasubunit Interactions during the Formation of RNA Polymerase Assembly Intermediate

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