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S Borukhov

Publications and source records attributed to S Borukhov.

17 recordsLinked to original sources
PubMed2000

The functional role of basic patch, a structural element of Escherichia coli transcript cleavage factors GreA and GreB.

The transcript cleavage factors GreA and GreB of Escherichia coli are involved in the regulation of transcription elongation. The surface charge distribution analysis of their three-dimensional structures revealed that the N-terminal domains of GreA and GreB contain a small and large basic "patch," respectively. To elucidate the functional role of basic patch, mutant Gre proteins were engineered in which the size and charge distribution of basic patch were modified and characterized biochemically. We found that Gre mutants lacking basic patch or carrying basic patch of decreased size bind to RNA polymerase and induce transcript cleavage reaction in minimally backtracked ternary elongation complex (TEC) with the same efficiency as the wild type factors. However, they exhibit substantially lower readthrough and cleavage activities toward extensively backtracked and arrested TECs and display decreased efficiency of photocross-linking to the RNA 3'-terminus. Unlike wild type factors, basic patch-less Gre mutants are unable to complement the thermosensitive phenotype of GreA(-):GreB(-) E. coli strain. The large basic patch is required but not sufficient for the induction of GreB-type cleavage reaction and for the cleavage of arrested TECs. Our results demonstrate that the basic patch residues are not directly involved in the induction of transcript cleavage reaction and suggest that the primary role of basic patch is to anchor the nascent RNA in TEC. These interactions are essential for the readthrough and antiarrest activities of Gre factors and, apparently, for their in vivo functions.

Amino Acid Sequence↗
PubMed1998

Visualization of the binding site for the transcript cleavage factor GreB on Escherichia coli RNA polymerase.

The structure of Escherichia coli core RNA polymerase (RNAP) complexed with the transcript cleavage factor GreB was determined from electron micrographs of negatively stained, flattened helical crystals. A binding assay was developed to establish that GreB was incorporated into the RNA polymerase crystals with high occupancy through interactions between the globular C-terminal domain and the RNA polymerase. Comparison of the core RNAP:GreB structure with the previously determined structure of core RNAP located the GreB binding site on one face of the RNA polymerase, next to but not in the 25 A-diameter channel of RNA polymerase.

Binding Sites↗
PubMed1998

Distinct functions of N and C-terminal domains of GreA, an Escherichia coli transcript cleavage factor.

The prokaryotic transcription factors GreA and GreB are involved in the regulation of transcript elongation by RNA polymerase (RNAP). Their known activities include suppression of transcription arrest, enhancement of transcription fidelity, and facilitation of the transition from abortive initiation to productive elongation. Presumably, Gre proteins exert their functions by altering the conformation of the enzyme in ternary elongation complexes (TEC) and inducing the cleavage of nascent RNA. GreA and GreB have a similar structural organization and consist of two domains: a C-terminal globular and an extended N-terminal coiled-coil domain. To investigate the functional roles of Gre domains, we expressed separately the N and C-terminal domains of GreA (NTD and CTD, respectively) and characterized their activities with in vitro assays. We demonstrate that the NTD possesses the residual transcript cleavage activity of the wild-type GreA. The CTD does not display any nucleolytic activity; however, it substantially increases the cleavage activity of the NTD. In contrast to NTD, the CTD competes with GreA and GreB for binding to RNAP and inhibits their transcript cleavage and antiarrest activities. Both domains individually and together inhibit transcription elongation. From these results we conclude that the NTD is responsible for the GreA induction of nucleolytic activity while the CTD determines the binding of GreA to RNAP. Both domains are required for full functional activity of GreA.

Binding Sites↗
PubMed1997

Domain organization of Escherichia coli transcript cleavage factors GreA and GreB.

The GreA and GreB proteins of Escherichia coli induce cleavage of the nascent transcript in ternary elongation complexes of RNA polymerase. Gre factors are presumed to have two biologically important and evolutionarily conserved functions: the suppression of elongation arrest and the enhancement of transcription fidelity. A three-dimensional structure of GreB was generated by homology modeling on the basis of the known crystal structure of GreA. Both factors display similar overall architecture and surface charge distribution, with characteristic C-terminal globular and N-terminal coiled-coil domains. One major difference between the two factors is the "basic patch" on the surface of the coiled-coil domain, which is much larger in GreB than in GreA. In both proteins, a site near the basic patch cross-links to the 3' terminus of RNA in the ternary transcription complex. GreA/GreB hybrid molecules were constructed by genetic engineering in which the N-terminal domain of one protein was fused to the C-terminal domain of the other. In the hybrid molecules, both the coiled-coil and the globular domains contribute to specific binding of Gre factors to RNA polymerase, whereas the antiarrest activity and the GreA or GreB specificity of transcript cleavage is determined by the N-terminal domain. These results implicate the basic patch of the N-terminal coiled-coil domain as an important functional element responsible for the interactions with nascent transcript and determining the size of the RNA fragment to be excised during the course of the cleavage reaction.

Amino Acid Sequence↗
PubMed1995

Intrinsic transcript cleavage activity of RNA polymerase.

The GreA and GreB transcript cleavage factors of Escherichia coli suppress elongation arrest and may have a proofreading role in transcription. With the use of E. coli greA-greB- mutant, RNA polymerase is demonstrated to possess substantial intrinsic transcript cleavage activity. Mildly alkaline pH mimics the effect of the Gre proteins by inducing transcript cleavage in ternary complexes and antagonizing elongation arrest through a cleavage-and-restart reaction. Thus, transcript cleavage constitutes the second enzymological activity of RNA polymerase along with polymerization/pyrophosphorolysis of RNA, whereas the Gre proteins merely enhance this intrinsic property.

Bacterial Proteins↗
PubMed1995

Crystal structure of the GreA transcript cleavage factor from Escherichia coli.

Transcription elongation factors stimulate the activity of DNA-dependent RNA polymerases by increasing the overall elongation rate and the completion of RNA chains. One group of such factors, which includes Escherichia coli GreA, GreB and eukaryotic SII (TFIIS), acts by inducing hydrolytic cleavage of the transcript within the RNA polymerase, followed by release of the 3'-terminal fragment. Here we report the crystal structure of GreA at 2.2 A resolution. The structure contains an amino-terminal domain consisting of an antiparallel alpha-helical coiled-coil dimer which extends into solution, reminiscent of the coiled coil in seryl-tRNA synthetases. A site near the tip of the coiled-coil 'finger' plays a direct role in the transcript cleavage reaction by contacting the 3'-end of the transcript. The structure exhibits an unusual asymmetric charge distribution which indicates the manner in which GreA interacts with the RNA polymerase elongation complex.

Amino Acid Sequence↗
PubMed1994

Crystallization of GreA, a transcript cleavage factor from Escherichia coli.

GreA is a 17.6 kDa protein from Escherichia coli that induces cleavage of the nascent transcript in the elongating complex of RNA polymerase, followed by release of the 3'-terminal fragment. Crystals of GreA have been obtained from polyethylene glycol 4000, 2-propanol and sodium citrate, pH 5.6 and have been propagated by a novel seeding procedure. The crystals diffract beyond 2 A resolution and belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 101.7 A, b = 42.22 A, c = 40.05 A and with one molecule in the asymmetric unit.

Bacterial Proteins↗
PubMed1993

Two modes of transcription initiation in vitro at the rrnB P1 promoter of Escherichia coli.

The rrnB P1 promoter of Escherichia coli (starting sequence C-4-A-3-C-2-C-1-A+1-C+2-U+3-G+4) forms a binary complex with RNA polymerase that is highly unstable and requires the presence of transcription substrates ATP and CTP for stabilizing the enzyme-DNA association (Gourse, R. L. (1988) Nucleic Acids Res. 16, 9789-9809). We show that in the absence of UTP and GTP the stabilization is accomplished by short RNA oligomers synthesized in an unusual "-3-->" mode whereby the primer initiated at the +1 site presumably slips back by three nucleotides into the -3 site and is then extended yielding stable ternary complexes. By contrast, short oligomers initiated in the conventional "+1-->" mode without slippage do not exert the stabilization effect and are readily aborted from the promoter complex. The stable -3-->ternary complexes carry sigma factor but otherwise resemble elongation complexes in their high salt stability and in the fact that they are formed with a mutant RNA polymerase deficient in promoter binding. A model is proposed explaining the stability of the -3-->ternary complexes by RNA slipping into a putative "tight RNA binding site" in RNA polymerase which is normally occupied by RNA during elongation.

Cold Temperature↗
PubMed1993

Transcript cleavage factors from E. coli.

Two transcription elongation factors (GreA and GreB) related in primary sequence were isolated from E. coli. Each factor induced cleavage of the nascent transcript in artificially halted elongation complexes followed by the loss of the 3' proximal fragment and resumption of elongation from the new 3' terminus. GreA induced cleavages 2 or 3 nt behind the terminus while GreB released longer oligonucleotides up to 9 nt in length. The pattern of cleavages characteristically changed as the transcription complex advanced, supporting the "inchworm" model of RNA polymerase propagation. In addition to attacking artificially halted complexes, both factors antagonized the action of natural elongation-arresting sites that occasionally trap the advancing complex. GreB rescued the arrested complexes via the transcript cleavage and restart pathway while GreA acted by an unknown mechanism, preventing the arrest only if added before the polymerase reached the arresting site.

Amino Acid Sequence↗
PubMed1993

Recombinant Escherichia coli RNA polymerase: purification of individually overexpressed subunits and in vitro assembly.

New improved methods were developed for the purification to apparent homogeneity of alpha, beta, beta', and sigma subunits of Escherichia coli RNA polymerase (RNAP) from corresponding overproducing strains. The purified subunits were assembled into enzymatically active RNAP holoenzyme (alpha 2 beta beta' sigma) using the optimal subunit molar ratio (alpha:beta:beta':sigma = 2:8:4:1) at a total protein concentration of 0.5 mg/ml. The presence of sigma subunit and 10 microM ZnCl2 in the reconstitution mixture increased the yield of RNAP approximately 4 times. The assembled RNA polymerase was purified by two successive chromatographic steps using size-exclusion Superose 6 and anion exchange Mono Q FPLC columns, which resulted in the electrophoretically homogeneous holoenzyme with overall yield of 56%. The specific activity of the recombinant RNAP estimated by the standard T4 transcription assay was 6.5 nmol of [3H]UTP incorporated into acid-insoluble RNA product per microgram of RNAP per 1 h.

Bacterial Proteins↗
PubMed1992

GreA protein: a transcription elongation factor from Escherichia coli.

A protein identified as the 158-amino acid product of the greA gene was isolated from Escherichia coli. When added to a halted ternary transcription complex, the GreA protein induced cleavage and removal of the 3' proximal dinucleotide from the nascent RNA. The new 3' terminus generated by the cleavage could be extended into longer transcripts. GreA-mediated cleavage of a transcript appears to permit a ternary complex to resume transcription from a state of indefinite elongation arrest induced by a specific DNA site. The GreA protein tended to interact with RNA polymerase during purification and recycled between RNA polymerase molecules in the course of the in vitro cleavage reaction. Similar biochemical activities have been reported in eukaryotic RNA polymerases, indicating that transcript cleavage and restart of elongation may be a general transcriptional mechanism.

Bacterial Proteins↗
PubMed1992

Dissection of the beta subunit in the Escherichia coli RNA polymerase into domains by proteolytic cleavage.

The 1342 amino acid long beta subunit of Escherichia coli RNA polymerase includes a dispensable region (residues 940-1040) that is absent in homologous RNA polymerase subunits from chloroplasts, eukaryotes, and archaebacteria (Borukhov, S., Severinov, K., Kashlev, M., Lebedev, A., Bass, I., Rowland, G. C., Lim, P.-P., Glass, R. E., Nikiforov, V., and Goldfarb, A. (1991) J. Biol. Chem. 266, 23921-23926). Genetic disruption of this region by in-frame deletion or insertion sensitizes the beta subunit in assembled RNA polymerase molecules to attack by trypsin. We demonstrate that RNA polymerase with the beta polypeptide cleaved in the dispensable region retains normal in vitro activity. Moreover, the RNA polymerase activity is completely restored after denaturation and reconstitution of the enzyme carrying cleaved beta subunit indicating that its carboxyl- and amino-terminal parts fold and assemble into RNA polymerase as separate entities.

Amino Acid Sequence↗
PubMed1991

Mapping of a contact for the RNA 3' terminus in the largest subunit of RNA polymerase.

Stalled elongation complexes of Escherichia coli RNA polymerase were prepared carrying the photo-cross-linkable 8-azido derivative of adenine at the 3'-terminus of the nascent RNA chain. Ultraviolet irradiation of such complexes resulted in the cross-linking of radiolabeled RNA exclusively to the beta' subunit of RNA polymerase. The adduct was mapped between Met932 and Trp1020 in the linear sequence of the beta' polypeptide using specific chemical degradation of the cross-linked species.

Adenosine Monophosphate↗
PubMed1991

Mapping of trypsin cleavage and antibody-binding sites and delineation of a dispensable domain in the beta subunit of Escherichia coli RNA polymerase.

We have mapped principal sites in the Escherichia coli RNA polymerase molecule that are exposed to attack by trypsin under limited proteolysis conditions. The 1342-amino acid-long beta subunit is alternatively cleaved at Arg903 or Lys909. The cleavage occurs adjacent to a dispensable domain (residues 940-1040) that is absent in the homologous RNA polymerase subunits from chloroplasts, eukaryotes, and archaebacteria. In E. coli, this region can be disrupted with genetic deletions and insertions without the loss of RNA polymerase function. Insertion of 127 amino acids into this region introduces a new highly labile site for trypsin proteolysis. The dispensable domain carries the epitope for monoclonal antibody PYN-6 (near residue 1000), which can be used for anchoring the catalytically active enzyme on a solid support. We also report the identification of a secondary trypsin cleavage at Arg81 of the beta' subunit within a putative zinc-binding domain that is conserved in prokaryotes and chloroplasts.

Amino Acid Sequence↗
PubMed1991

A beta subunit mutation disrupting the catalytic function of Escherichia coli RNA polymerase.

The substitution of the evolutionarily conserved Glu-813 for lysine in the beta subunit of RNA polymerase (RNAP) causes a partial loss of function in the assembled RNAP. In the presence of the four ribonucleoside triphosphates, the mutant RNAP displayed a decreased frequency of promoter clearance and diminished elongation rate. Both defects could be compensated by raising the ribonucleoside triphosphate concentration. In the abortive initiation reaction limited by the incomplete set of ribonucleoside triphosphates, the mutant RNAP generated aberrant patterns of products indicative of their enhanced loss from the RNAP-promoter complex. A model is proposed, attributing the multiple effect of the mutation to the malfunctioning of the RNAP active center.

Amino Acid Sequence↗