Biomedical subjects

Nikolay Zenkin

Publications and source records attributed to Nikolay Zenkin.

10 recordsLinked to original sources

PubMed2006

Transcript-assisted transcriptional proofreading.

Fidelity of template-dependent nucleic acid synthesis is the main determinant of stable heredity and error-free gene expression. The mechanism (or mechanisms) ensuring fidelity of transcription by DNA-dependent RNA polymerases (RNAPs) is not fully understood. Here, we show that the 3' end-proximal nucleotide of the nascent transcript stimulates hydrolysis of the penultimate phosphodiester bond by providing active groups and coordination bonds to the RNAP active center. This stimulation is much higher in the case of misincorporated nucleotide. We show that during transcription elongation, the hydrolytic reaction stimulated by misincorporated nucleotides proofreads most of the misincorporation events and thus serves as an intrinsic mechanism of transcription fidelity.

Adenosine Monophosphate↗

PubMed2006

The mechanism of DNA replication primer synthesis by RNA polymerase.

RNA primers for DNA replication are usually synthesized by specialized enzymes, the primases. However, some replication systems have evolved to use cellular DNA-dependent RNA polymerase for primer synthesis. The main requirement for the replication primer, an exposed RNA 3' end annealed to the DNA template, is not compatible with known conformations of the transcription elongation complex, raising a question of how the priming is achieved. Here we show that a previously unrecognized kind of transcription complex is formed during RNA polymerase-catalysed synthesis of the M13 bacteriophage replication primer. The complex contains an overextended RNA-DNA hybrid bound in the RNA-polymerase trough that is normally occupied by downstream double-stranded DNA, thus leaving the 3' end of the RNA available for interaction with DNA polymerase. Transcription complexes with similar topology may prime the replication of other bacterial mobile elements and may regulate transcription elongation under conditions that favour the formation of an extended RNA-DNA hybrid.

Bacteriophage M13↗

PubMed2005

Structural basis of transcription inhibition by antibiotic streptolydigin.

Streptolydigin (Stl) is a potent inhibitor of bacterial RNA polymerases (RNAPs). The 2.4 A resolution structure of the Thermus thermophilus RNAP-Stl complex showed that, in full agreement with the available genetic data, the inhibitor binding site is located 20 A away from the RNAP active site and encompasses the bridge helix and the trigger loop, two elements that are considered to be crucial for RNAP catalytic center function. Structure-based biochemical experiments revealed additional determinants of Stl binding and demonstrated that Stl does not affect NTP substrate binding, DNA translocation, and phosphodiester bond formation. The RNAP-Stl complex structure, its comparison with the closely related substrate bound eukaryotic transcription elongation complexes, and biochemical analysis suggest an inhibitory mechanism in which Stl stabilizes catalytically inactive (preinsertion) substrate bound transcription intermediate, thereby blocking structural isomerization of RNAP to an active configuration. The results provide a basis for a design of new antibiotics utilizing the Stl-like mechanism.

Amino Acid Sequence↗

PubMed2005

Remodeling of the sigma70 subunit non-template DNA strand contacts during the final step of transcription initiation.

Transcription initiation in bacteria requires melting of approximately 13 bp of promoter DNA. The mechanism of the melting process is not fully understood. Escherichia coli RNA polymerase bearing a deletion of the beta subunit lobe I (amino acid residues 186-433) initiates melting of the -10 promoter element but cannot propagate the melting downstream, towards the transcription initiation start site (+1). However, in the presence of nucleotides, stable downstream melting is induced. Here, we studied lacUV5 promoter complexes formed by the mutant enzyme by cross-linking RNA polymerase subunits to single-stranded DNA in the transcription bubble. In the absence of NTPs, a contact between the sigma70 subunit and the non-template strand of the -10 promoter element was detected. This contact disappeared in the presence of NTPs. Instead, a new sigma70-DNA contact as well as stable beta' and beta subunit contacts with the non-template DNA downstream of the -10 promoter element were established. In terms of the two-step (upstream initiation/downstream propagation) model of promoter melting, our data suggest that beta lobe I induces the propagation of promoter melting by directing downstream promoter DNA duplex towards the downstream DNA-binding channel (beta' clamp). Establishment of downstream contacts leads to remodeling of upstream interactions between sigma70 and the -10 promoter element that might facilitate promoter escape and sigma release.

DNA, Bacterial↗

PubMed2005

Structural, functional, and genetic analysis of sorangicin inhibition of bacterial RNA polymerase.

A combined structural, functional, and genetic approach was used to investigate inhibition of bacterial RNA polymerase (RNAP) by sorangicin (Sor), a macrolide polyether antibiotic. Sor lacks chemical and structural similarity to the ansamycin rifampicin (Rif), an RNAP inhibitor widely used to treat tuberculosis. Nevertheless, structural analysis revealed Sor binds in the same RNAP beta subunit pocket as Rif, with almost complete overlap of RNAP binding determinants, and functional analysis revealed that both antibiotics inhibit transcription by directly blocking the path of the elongating transcript at a length of 2-3 nucleotides. Genetic analysis indicates that Rif binding is extremely sensitive to mutations expected to change the shape of the antibiotic binding pocket, while Sor is not. We suggest that conformational flexibility of Sor, in contrast to the rigid conformation of Rif, allows Sor to adapt to changes in the binding pocket. This has important implications for drug design against rapidly mutating targets.

Aminoglycosides↗

PubMed2004

Regulated communication between the upstream face of RNA polymerase and the beta' subunit jaw domain.

We used bacteriophage T7-encoded transcription inhibitor gene protein 2 (gp2) as a probe to study the contribution of the Escherichia coli RNA polymerase (RNAP) beta' subunit jaw domain--the site of gp2 binding--to activator and ATP hydrolysis-dependent open complex formation by the sigma(54)-RNAP. We show that, unlike sigma(70)-dependent transcription, activated transcription by sigma(54)-RNAP is resistant to gp2. In contrast, activator and ATP hydrolysis-independent transcription by sigma(54)-RNAP is highly sensitive to gp2. We provide evidence that an activator- and ATP hydrolysis-dependent conformational change involving the beta' jaw domain and promoter DNA is the basis for gp2-resistant transcription by sigma(54)-RNAP. Our results establish that accessory factors bound to the upstream face of the RNAP, communicate with the beta' jaw domain, and that such communication is subjected to regulation.

Bacterial Proteins↗

PubMed2004

Molecular mechanism of transcription inhibition by peptide antibiotic Microcin J25.

21 amino acid peptide Microcin J25 (MccJ25) inhibits transcription by bacterial RNA polymerase (RNAP). MccJ25-resistance mutations cluster in the RNAP secondary channel through which incoming NTP substrates are thought to reach the catalytic center and the 3' end of the nascent RNA is likely to thread in backtracked transcription complexes. The secondary channel also accepts transcript cleavage factors GreA and GreB. Here, we demonstrate that MccJ25 inhibits GreA/GreB-dependent transcript cleavage, impedes formation of backtracked complexes, and can be crosslinked to the 3'-end of the nascent RNA in elongation complexes. These results place the MccJ25 binding site within the secondary channel. Moreover, single-molecule assays reveal that MccJ25 binding to a transcribing RNAP temporarily stops transcript elongation but has no effect on the elongation velocity between pauses. Kinetic analysis of single-molecule data allows us to put forward a model of transcription inhibition by MccJ25 that envisions the complete occlusion of the secondary channel by bound inhibitor.

Anti-Bacterial Agents↗

PubMed2004

The sigma 70 subunit of RNA polymerase induces lacUV5 promoter-proximal pausing of transcription.

The sigma(70) subunit of Escherichia coli RNA polymerase (RNAP) is a transcription initiation factor that can also be associated with RNAP during elongation. We provide biochemical evidence that sigma(70) induces a transcription pause at the lacUV5 promoter after RNAP has synthesized a 17-nucleotide transcript. The sigma(70)-dependent pausing requires an interaction between sigma(70) and a part of the lac repressor operator sequence resembling a promoter -10 consensus. The polysaccharide heparin triggers the release of sigma(70) from the paused complexes, supporting the view that during the transition from initiation to elongation the interactions between sigma(70) and core RNAP are weakened. We propose that the binding and retention of sigma(70) in elongation complexes are stabilized by its ability to form contacts with DNA of the transcription bubble. In addition, we suggest that the sigma(70) subunit in the elongation complex may provide a target for regulation of gene expression.

Base Sequence↗

PubMed2004

The role of RNA polymerase sigma subunit in promoter-independent initiation of transcription.

In bacteria, initiation of transcription depends on the RNA polymerase sigma subunit, which brings catalytically proficient RNA polymerase core to promoters by binding to specific DNA elements located upstream of the transcription start point. Here, we study sigma-dependent synthesis of a transcript that is used to prime replication of the single-stranded genome of bacteriophage M13. We show that, in this system, sigma plays no role in DNA recognition, which is accomplished solely through RNA polymerase core interaction with DNA downstream of the transcription start point. However, sigma is required for full-sized transcript synthesis by allowing RNA polymerase core to escape into productive elongation. RNA polymerase sigma may play a similar role during replication primer synthesis in other bacterial mobile elements whose life cycle involves a single-stranded DNA stage.

Base Sequence↗

PubMed2004

A new Bacillus cereus DNA-binding protein, HlyIIR, negatively regulates expression of B. cereus haemolysin II.

Haemolysin II, HlyII, is one of several cytotoxic proteins produced by Bacillus cereus, an opportunistic human pathogen that causes food poisoning. The hlyII gene confers haemolytic activity to Escherichia coli cells. Here a new B. cereus gene, hlyIIR, which is located immediately downstream of hlyII and regulates hlyII expression, is reported. The deduced amino acid sequence of HlyIIR is similar to prokaryotic DNA-binding transcriptional regulators of the TetR/AcrA family. Measurements of haemolytic activity levels and of hlyII promoter activity levels using gene fusions and primer-extension assays demonstrated that, in E. coli, hlyII transcription decreased in the presence of hlyIIR. Recombinant HlyIIR binds to a 22 bp inverted DNA repeat centred 48 bp upstream of the hlyII promoter transcription initiation point. In vitro transcription studies showed that HlyIIR inhibits transcription from the hlyII promoter by binding to the 22 bp repeat and RNA polymerase, and by decreasing the formation of the catalytically competent open promoter complex.

Artificial Gene Fusion↗