Petr V. Sergiev

Learn More
Two photoreactive diazirine derivatives of uridine were used to study contacts between 5S rRNA and 23 rRNA in situ in Escherichia coli ribosomes. 2'-Amino-2'-deoxy-uridine or 5-methyleneaminouridine were introduced into 5S rRNA by T7 transcription. After incorporation of these uridine analogues into the transcript their amino groups were modified with(More)
The proximity of loop D of 5 S rRNA to two regions of 23 S rRNA, domain II involved in translocation and domain V involved in peptide bond formation, is known from previous cross-linking experiments. Here, we have used site-directed mutagenesis and chemical probing to further define these contacts and possible sites of communication between 5 S and 23 S(More)
Two new photoreactive nucleotide derivatives have been applied in site-directed crosslinking studies with mRNA analogues. 6-Thioguanosine triphosphate or 5-methyleneaminouridine triphosphate was incorporated into mRNA analogues by T7 transcription; after transcription, the 5-methyleneaminouridine residues were converted to a diazirine derivative. mRNA(More)
Radioactively labeled 4.5S RNA containing statistically distributed 4-thiouridine residues in place of normal uridine was prepared by T7 transcription. The ability of this modified 4.5S RNA to form a complex with the protein Ffh was demonstrated by a gel shift assay. The modified 4.5S RNA, with or without Ffh, was added to Escherichia coli ribosomes under(More)
Regulation of gene expression at the level of translation accounts for up to three orders of magnitude in its efficiency. We systematically compared the impact of several mRNA features on translation initiation at the first gene in an operon with those for the second gene. Experiments were done in a system with internal control based on dual cerulean and(More)
We demonstrate that the antibiotic amicoumacin A (AMI) is a potent inhibitor of protein synthesis. Resistance mutations in helix 24 of the 16S rRNA mapped the AMI binding site to the small ribosomal subunit. The crystal structure of bacterial ribosome in complex with AMI solved at 2.4 Å resolution revealed that the antibiotic makes contacts with universally(More)
Elongation factors EF-G and EF-Tu are structural homologues and share near-identical binding sites on the ribosome, which encompass the GTPase-associated centre (GAC) and the sarcin-ricin loop (SRL). The SRL is fixed structure in the ribosome and contacts elongation factors in the vicinity of their GTP-binding site. In contrast, the GAC is mobile and we(More)
A method for preparation of Escherichia coli ribosomes carrying lethal mutations in 23 S rRNA was developed. The method is based on the site-directed incorporation of a streptavidin binding tag into functionally neutral sites of the 23 S rRNA and subsequent affinity chromatography. It was tested with ribosomes mutated at the 23 S rRNA position 2655 (the(More)
Translocation catalyzed by elongation factor G occurs after the peptidyltransferase reaction on the large ribosomal subunit. Deacylated tRNA in the P-site stimulates multiple turnover GTPase activity of EF-G. We suggest that the allosteric signal from the peptidyltransferase center that activates EF-G may involve the alteration in the conformation of(More)
Ribosomes synthesize proteins according to the information encoded in mRNA. During this process, both the incoming amino acid and the nascent peptide are bound to tRNA molecules. Three binding sites for tRNA in the ribosome are known: the A-site for aminoacyl-tRNA, the P-site for peptidyl-tRNA and the E-site for the deacylated tRNA leaving the ribosome.(More)