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Marina V Rodnina

Publications and source records attributed to Marina V Rodnina.

At least 19 recordsLinked to original sources
PubMed2007

Codon reading by tRNAAla with modified uridine in the wobble position.

tRNAs reading four-codon families often have a modified uridine, cmo(5)U(34), at the wobble position of the anticodon. Here, we examine the effects on the decoding mechanism of a cmo(5)U modification in tRNA(1B)(Ala), anticodon C(36)G(35)cmo(5)U(34). tRNA(1B)(Ala) reads its cognate codons in a manner that is very similar to that of tRNA(Phe). As Ala codons are GC rich and Phe codons AU rich, this similarity suggests a uniform decoding mechanism that is independent of the GC content of the codon-anticodon duplex or the identity of the tRNA. The presence of cmo(5)U at the wobble position of tRNA(1B)(Ala) permits fairly efficient reading of non-Watson-Crick and nonwobble bases in the third codon position, e.g., the GCC codon. The ribosome accepts the C-cmo(5)U pair as an almost-correct base pair, unlike third-position mismatches, which lead to the incorporation of incorrect amino acids and are efficiently rejected.

Base Sequence↗
PubMed2006

How ribosomes make peptide bonds.

Ribosomes are molecular machines that synthesize proteins in the cell. Recent biochemical analyses and high-resolution crystal structures of the bacterial ribosome have shown that the active site for the formation of peptide bonds--the peptidyl-transferase center--is composed solely of rRNA. Thus, the ribosome is the largest known RNA catalyst and the only natural ribozyme that has a synthetic activity. The ribosome employs entropic catalysis to accelerate peptide-bond formation by positioning substrates, reorganizing water in the active site and providing an electrostatic network that stabilizes reaction intermediates. Proton transfer during the reaction seems to be promoted by a concerted shuttle mechanism that involves ribose hydroxyl groups on the tRNA substrate.

Binding Sites↗
PubMed2006

Colicins and their potential in cancer treatment.

Colicins are a family of antibacterial cytotoxins produced by Escherichia coli and released into the environment to reduce competition from other bacterial strains. Colicins kill the target cell by a variety of effects that include depolarisation of the cytoplasmic membrane, a non-specific DNase activity, a highly specific RNase activity or by inhibition of murein synthesis. This review summarises some important findings that implicate colicins as potential anti-tumor agents. Colicins appear to inhibit proliferation of tumor cell lines in a colicin-type--and cell line-dependent fashion and are more toxic to tumor cells than to normal cells within the body. This opens a potential for using bacterial colicins in combating cancer and raises a number of questions concerning the mechanism of action of colicins in targeting tumor cells, their specificity and applicability as anti-tumor therapeutics.

Animals↗
PubMed2006

Delayed release of inorganic phosphate from elongation factor Tu following GTP hydrolysis on the ribosome.

The dissociation of inorganic phosphate (P(i)) following GTP hydrolysis is a key step determining the functional state of many GTPases. Here, the timing of P(i) release from elongation factor Tu (EF-Tu) and its implications for the function of EF-Tu on the ribosome were studied by rapid kinetic techniques. It was found that P(i) release from EF-Tu is >20-fold slower than GTP cleavage and limits the rate of the conformational switch of EF-Tu from the GTP- to the GDP-bound form. The point mutation Gly94Ala in the switch 2 region of EF-Tu abolished the delay in P(i) release, suggesting that P(i) release is controlled by the mobility of the switch 2 region with Gly94 acting as a pivot. The rate of P(i) release or the conformational switch of EF-Tu does not affect the selection of aminoacyl-tRNA on the ribosome. Rather, the slow P(i) release may be a consequence of the tight interaction of the switch regions of EF-Tu with the gamma-phosphate and the ribosome in the GTPase activated state of the factor.

Escherichia coli↗
PubMed2006

Kinetic analysis of interaction of eukaryotic release factor 3 with guanine nucleotides.

Eukaryotic translation termination is mediated by two release factors: eRF1 recognizes stop codons and triggers peptidyl-tRNA hydrolysis, whereas eRF3 accelerates this process in a GTP-dependent manner. Here we report kinetic analysis of guanine nucleotide binding to eRF3 performed by fluorescence stopped-flow technique using GTP/GDP derivatives carrying the fluorescent methylanthraniloyl (mant-) group, as well as thermodynamic analysis of eRF3 binding to unlabeled guanine nucleotides. Whereas the kinetics of eRF3 binding to mant-GDP is consistent with a one-step binding model, the double-exponential transients of eRF3 binding to mant-GTP indicate a two-step binding mechanism, in which the initial eRF3.mant-GTP complex undergoes subsequent conformational change. The affinity of eRF3 for GTP (K(d), approximately 70 microM) is about 70-fold lower than for GDP (K(d), approximately 1 microM) and both nucleotides dissociate rapidly from eRF3 (k(-1)(mant-GDP) approximately 2.4 s(-1); k(-2)(mant-GTP) approximately 3.3 s(-1)). Whereas not influencing eRF3 binding to GDP, association of eRF3 with eRF1 at physiological Mg(2+) concentrations specifically changes the kinetics of eRF3/mant-GTP interaction and stabilizes eRF3.GTP binding by two orders of magnitude (K(d) approximately 0.7 microM) due to lowering of the dissociation rate constant approximately 24-fold (k(-1)(mant-GTP) approximately 0.14s(-1) approximately 0.14 s(-1)). Thus, eRF1 acts as a GTP dissociation inhibitor (TDI) for eRF3, promoting efficient ribosomal recruitment of its GTP-bound form. 80 S ribosomes did not influence guanine nucleotide binding/exchange on the eRF1 x eRF3 complex. Guanine nucleotide binding and exchange on eRF3, which therefore depends on stimulation by eRF1, is entirely different from that on prokaryotic RF3 and unusual among GTPases.

Animals↗
PubMed2006

Involvement of helix 34 of 16 S rRNA in decoding and translocation on the ribosome.

Helix 34 of 16 S rRNA is located in the head of the 30 S ribosomal subunit close to the decoding center and has been invoked in a number of ribosome functions. In the present work, we have studied the effects of mutations in helix 34 both in vivo and in vitro. Several nucleotides in helix 34 that are either highly conserved or form important tertiary contacts in 16 S rRNA (U961, C1109, A1191, and A1201) were mutated, and the mutant ribosomes were expressed in the Escherichia coli MC250 Delta7 strain that lacks all seven chromosomal rRNA operons. Mutations at positions A1191 and U961 reduced the efficiency of subunit association and resulted in structural rearrangements in helix 27 (position 908) and helix 31 (position 974) of 16 S rRNA. All mutants exhibited increased levels of frameshifting and nonsense readthrough. The effects on frameshifting were specific in that -1 frameshifting was enhanced with mutant A1191G and +1 frameshifting with the other mutants. Mutations of A1191 moderately (approximately 2-fold) inhibited tRNA translocation. No significant effects were found on efficiency and rate of initiation, misreading of sense codons, or binding of tRNA to the E site. The data indicate that helix 34 is involved in controlling the maintenance of the reading frame and in tRNA translocation.

Escherichia coli↗
PubMed2006

The nucleotide-binding site of bacterial translation initiation factor 2 (IF2) as a metabolic sensor.

Translational initiation factor 2 (IF2) is a guanine nucleotide-binding protein that can bind guanosine 3',5'-(bis) diphosphate (ppGpp), an alarmone involved in stringent response in bacteria. In cells growing under optimal conditions, the GTP concentration is very high, and that of ppGpp very low. However, under stress conditions, the GTP concentration may decline by as much as 50%, and that of ppGpp can attain levels comparable to those of GTP. Here we show that IF2 binds ppGpp at the same nucleotide-binding site and with similar affinity as GTP. Thus, GTP and the alarmone ppGpp can be considered two alternative physiologically relevant IF2 ligands. ppGpp interferes with IF2-dependent initiation complex formation, severely inhibits initiation dipeptide formation, and blocks the initiation step of translation. Our data suggest that IF2 has the properties of a cellular metabolic sensor and regulator that oscillates between an active GTP-bound form under conditions allowing active protein syntheses and an inactive ppGpp-bound form when shortage of nutrients would be detrimental, if not accompanied by slackening of this synthesis.

Binding Sites↗
PubMed2006

Role and timing of GTP binding and hydrolysis during EF-G-dependent tRNA translocation on the ribosome.

The translocation of tRNA and mRNA through the ribosome is promoted by elongation factor G (EF-G), a GTPase that hydrolyzes GTP during the reaction. Recently, it was reported that, in contrast to previous observations, the affinity of EF-G was much weaker for GTP than for GDP and that ribosome-catalyzed GDP-GTP exchange would be required for translocation [Zavialov AV, Hauryliuk VV, Ehrenberg M (2005) J Biol 4:9]. We have reinvestigated GTP/GDP binding and show that EF-G binds GTP and GDP with affinities in the 20 to 40 microM range (37 degrees C), in accordance with earlier reports. Furthermore, GDP exchange, which is extremely rapid on unbound EF-G, is retarded, rather than accelerated, on the ribosome, which, therefore, is not a nucleotide-exchange factor for EF-G. The EF-G.GDPNP complex, which is very labile, is stabilized 30,000-fold by binding to the ribosome. These findings, together with earlier kinetic results, reveal that EF-G enters the pretranslocation ribosome in the GTP-bound form and indicate that, upon ribosome-complex formation, the nucleotide-binding pocket of EF-G is closed, presumably in conjunction with GTPase activation. GTP hydrolysis is required for rapid tRNA-mRNA movement, and P(i) release induces further rearrangements of both EF-G and the ribosome that are required for EF-G turnover.

Dissociative Disorders↗
PubMed2006

Mechanism of peptide bond formation on the ribosome.

Peptide bond formation is the fundamental reaction of ribosomal protein synthesis. The ribosome's active site--the peptidyl transferase center--is composed of rRNA, and thus the ribosome is the largest known RNA catalyst. The ribosome accelerates peptide bond formation by 10(7)-fold relative to the uncatalyzed reaction. Recent progress of structural, biochemical and computational approaches has provided a fairly detailed picture of the catalytic mechanisms employed by the ribosome. Energetically, catalysis is entirely entropic, indicating an important role of solvent reorganization, substrate positioning, and/or orientation of the reacting groups within the active site. The ribosome provides a pre-organized network of electrostatic interactions that stabilize the transition state and facilitate proton shuttling involving ribose hydroxyl groups of tRNA. The catalytic mechanism employed by the ribosome suggests how ancient RNA-world enzymes may have functioned.

Binding Sites↗
PubMed2006

Rapid peptide bond formation on isolated 50S ribosomal subunits.

The catalytic site of the ribosome, the peptidyl transferase centre, is located on the large (50S in bacteria) ribosomal subunit. On the basis of results obtained with small substrate analogues, isolated 50S subunits seem to be less active in peptide bond formation than 70S ribosomes by several orders of magnitude, suggesting that the reaction mechanisms on 50S subunits and 70S ribosomes may be different. Here we show that with full-size fMet-tRNA(fMet) and puromycin or C-puromycin as peptide donor and acceptor substrates, respectively, the reaction proceeds as rapidly on 50S subunits as on 70S ribosomes, indicating that the intrinsic activity of 50S subunits is not different from that of 70S ribosomes. The faster reaction on 50S subunits with fMet-tRNA(fMet), compared with oligonucleotide substrate analogues, suggests that full-size transfer RNA in the P site is important for maintaining the active conformation of the peptidyl transferase centre.

Binding Sites↗
PubMed2006

The importance of P-loop and domain movements in EF-Tu for guanine nucleotide exchange.

Elongation factor Ts (EF-Ts) is the guanine nucleotide exchange factor for elongation factor Tu (EF-Tu). An important feature of the nucleotide exchange is the structural rearrangement of EF-Tu in the EF-Tu.EF-Ts complex caused by insertion of Phe-81 of EF-Ts between His-84 and His-118 of EF-Tu. In this study, the contribution of His-118 to nucleotide release was studied by pre-steady state kinetic analysis of nucleotide exchange in EF-Tu mutants in which His-118 was replaced by Ala or Glu. Intrinsic as well as EF-Ts-catalyzed release of GDP/GTP was affected by the mutations, resulting in an approximately 10-fold faster spontaneous nucleotide release and a 10-50-fold slower EF-Ts-catalyzed nucleotide release. The effects are attributed to the interference of the mutations with the EF-Ts-induced movements of the P-loop of EF-Tu and changes at the domain 1/3 interface, leading to the release of the beta-phosphate group of GTP/GDP. The K(d) for GTP is increased by more than 40 times when His-118 is replaced with Glu, which may explain the inhibition by His-118 mutations of aminoacyl-tRNA binding to EF-Tu. The mutations had no effect on EF-Tu-dependent delivery of aminoacyl-tRNA to the ribosome.

Catalysis↗
PubMed2006

Peptide bond formation does not involve acid-base catalysis by ribosomal residues.

Ribosomes catalyze the formation of peptide bonds between aminoacyl esters of transfer RNAs within a catalytic center composed of ribosomal RNA only. Here we show that the reaction of P-site formylmethionine (fMet)-tRNA(fMet) with a modified A-site tRNA substrate, Phelac-tRNA(Phe), in which the nucleophilic amino group is replaced with a hydroxyl group, does not show the pH dependence observed with small substrate analogs such as puromycin and hydroxypuromycin. This indicates that acid-base catalysis by ribosomal residues is not important in the reaction with the full-size substrate. Rather, the ribosome catalyzes peptide bond formation by positioning the tRNAs, or their 3' termini, through interactions with rRNA that induce and/or stabilize a pH-insensitive conformation of the active site and provide a preorganized environment facilitating the reaction. The rate of peptide bond formation with unmodified Phe-tRNA(Phe) is estimated to be >300 s(-1).

Acids↗
PubMed2006

A uniform response to mismatches in codon-anticodon complexes ensures ribosomal fidelity.

Ribosomes take an active part in aminoacyl-tRNA selection by distinguishing correct and incorrect codon-anticodon pairs. Correct codon-anticodon complexes are recognized by a network of ribosome contacts that are specific for each position of the codon-anticodon duplex and involve A-minor RNA interactions. Here, we show by kinetic analysis that single mismatches at any position of the codon-anticodon complex result in slower forward reactions and a uniformly 1000-fold faster dissociation of the tRNA from the ribosome. This suggests that high-fidelity tRNA selection is achieved by a conformational switch of the decoding site between accepting and rejecting modes, regardless of the thermodynamic stability of the respective codon-anticodon complexes or their docking partners at the decoding site. The forward reactions on mismatched codons were particularly sensitive to the disruption of the A-minor interactions with 16S rRNA and determined the variations in the misreading efficiency of near-cognate codons.

Animals↗
PubMed2005

Control of phosphate release from elongation factor G by ribosomal protein L7/12.

Ribosomal protein L7/12 is crucial for the function of elongation factor G (EF-G) on the ribosome. Here, we report the localization of a site in the C-terminal domain (CTD) of L7/12 that is critical for the interaction with EF-G. Single conserved surface amino acids were replaced in the CTD of L7/12. Whereas mutations in helices 5 and 6 had no effect, replacements of V66, I69, K70, and R73 in helix 4 increased the Michaelis constant (KM) of EF-G.GTP for the ribosome, suggesting an involvement of these residues in EF-G binding. The mutations did not appreciably affect rapid single-round GTP hydrolysis and had no effect on tRNA translocation on the ribosome. In contrast, the release of inorganic phosphate (Pi) from ribosome-bound EF-G.GDP.Pi was strongly inhibited and became rate-limiting for the turnover of EF-G. The control of Pi release by interactions between EF-G and L7/12 appears to be important for maintaining the conformational coupling between EF-G and the ribosome for translocation and for timing the dissociation of the factor from the ribosome.

Crystallography, X-Ray↗
PubMed2005

Structural and functional investigation of a putative archaeal selenocysteine synthase.

Bacterial selenocysteine synthase converts seryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec) for selenoprotein biosynthesis. The identity of this enzyme in archaea and eukaryotes is unknown. On the basis of sequence similarity, a conserved open reading frame has been annotated as a selenocysteine synthase gene in archaeal genomes. We have determined the crystal structure of the corresponding protein from Methanococcus jannaschii, MJ0158. The protein was found to be dimeric with a distinctive domain arrangement and an exposed active site, built from residues of the large domain of one protomer alone. The shape of the dimer is reminiscent of a substructure of the decameric Escherichia coli selenocysteine synthase seen in electron microscopic projections. However, biochemical analyses demonstrated that MJ0158 lacked affinity for E. coli seryl-tRNA(Sec) or M. jannaschii seryl-tRNA(Sec), and neither substrate was directly converted to selenocysteinyl-tRNA(Sec) by MJ0158 when supplied with selenophosphate. We then tested a hypothetical M. jannaschii O-phosphoseryl-tRNA(Sec) kinase and demonstrated that the enzyme converts seryl-tRNA(Sec) to O-phosphoseryl-tRNA(Sec) that could constitute an activated intermediate for selenocysteinyl-tRNA(Sec) production. MJ0158 also failed to convert O-phosphoseryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec). In contrast, both archaeal and bacterial seryl-tRNA synthetases were able to charge both archaeal and bacterial tRNA(Sec) with serine, and E. coli selenocysteine synthase converted both types of seryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec). These findings demonstrate that a number of factors from the selenoprotein biosynthesis machineries are cross-reactive between the bacterial and the archaeal systems but that MJ0158 either does not encode a selenocysteine synthase or requires additional factors for activity.

Amino Acid Sequence↗
PubMed2005

Essential mechanisms in the catalysis of peptide bond formation on the ribosome.

Peptide bond formation is the main catalytic function of the ribosome. The mechanism of catalysis is presumed to be highly conserved in all organisms. We tested the conservation by comparing mechanistic features of the peptidyl transfer reaction on ribosomes from Escherichia coli and the Gram-positive bacterium Mycobacterium smegmatis. In both cases, the major contribution to catalysis was the lowering of the activation entropy. The rate of peptide bond formation was pH independent with the natural substrate, amino-acyl-tRNA, but was slowed down 200-fold with decreasing pH when puromycin was used as a substrate analog. Mutation of the conserved base A2451 of 23 S rRNA to U did not abolish the pH dependence of the reaction with puromycin in M. smegmatis, suggesting that A2451 did not confer the pH dependence. However, the A2451U mutation alters the structure of the peptidyl transferase center and changes the pattern of pH-dependent rearrangements, as probed by chemical modification of 23 S rRNA. A2451 seems to function as a pivot point in ordering the structure of the peptidyl transferase center rather than taking part in chemical catalysis.

Alleles↗
PubMed2005

Conformations of the signal recognition particle protein Ffh from Escherichia coli as determined by FRET.

The signal recognition particle (SRP) initiates the co-translational targeting of proteins to the plasma membrane in bacteria by binding to the N-terminal signal sequence emerging from the translating ribosome. SRP in Escherichia coli is composed of one protein, Ffh, and 4.5S RNA. In the present work, we probe the structure of Ffh alone and in the complex with 4.5S RNA by measuring distances between different positions within Ffh and between Ffh and 4.5S RNA by fluorescence resonance energy transfer (FRET). According to the FRET distances, NG and M domains in free Ffh are in close contact, as in the A/A arrangement in the crystal structure of Ffh from Thermus aquaticus, in agreement with the formation of a crosslink between cysteine residues at two critical positions in the G and M domains. Upon Ffh binding to 4.5S RNA or a 61 nucleotide fragment comprising internal loops A-C, the G and M domains move apart to assume a more open conformation, as indicated by changes of FRET distances. The movement is smaller when Ffh binds to a 49 nucleotide fragment of 4.5S RNA comprising only internal loops A and B, i.e. lacking the binding site of the NG domain. The FRET results suggest that in the SRP complex 4.5S RNA is present in a bent, rather than extended, conformation. The domain rearrangement of Ffh that takes place upon formation of the SRP is probably important for subsequent steps of membrane targeting, including interactions with the translating ribosome and the SRP receptor.

Anisotropy↗