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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)
Among 4.5 thousand nucleotides of Escherichia coli ribosome 36 are modified. These nucleotides are clustered in the functional centers of ribosome, particularly on the interface of large and small subunits. Nucleotide m(2)G1835 located on the 50S side of intersubunit bridge cluster B2 is modified by N2-methyltransferase RlmG. By means of isothermal(More)
Chemical landscape of natural RNA species is decorated with the large number of modified nucleosides. Some of those could easily be detected by reverse transcription, while others permit only high-performance liquid chromatography or mass-spectrometry detection. Presence of m(6)A nucleoside at a particular position of long RNA molecule is challenging to(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)
The Database of Ribosomal Cross-links (DRC) provides a complete collection of all the published data produced by cross-linking studies on the Escherichia coli ribosome, as well as on its components and functional ligands. The DRC currently includes data on 986 cross-links from >100 research papers, yielded by >40 different reagents. For each cross-link,(More)
During protein synthesis, aminoacyl-tRNA (aa-tRNA) and release factors 1 and 2 (RF1 and RF2) have to bind at the catalytic center of the ribosome on the 50S subunit where they take part in peptide bond formation or peptidyl-tRNA hydrolysis, respectively. Computer simulations of aa-tRNA movement into the catalytic site (accommodation) suggested that three(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)
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)
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)