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At least 685 records · Page 38Linked to original sources
PubMed2006

Unfolding of mRNA secondary structure by the bacterial translation initiation complex.

Translation initiation is a key step for regulating the level of numerous proteins within the cell. In bacteria, the 30S initiation complex directly binds to the translation initiation region (TIR) of the mRNA. How the ribosomal 30S subunit assembles on highly structured TIR is not known. Using fluorescence-based experiments, we assayed 12 different mRNAs that form secondary structures with various stabilities and contain Shine-Dalgarno (SD) sequences of different strengths. A strong correlation was observed between the stability of the mRNA structure and the association and dissociation rate constants. Interestingly, in the presence of initiation factors and initiator tRNA, the association kinetics of structured mRNAs showed two distinct phases. The second phase was found to be important for unfolding structured mRNAs to form a stable 30S initiation complex. We show that unfolding of structured mRNAs requires a SD sequence, the start codon, fMet-tRNA(fMet), and the GTP bound form of initiation factor 2 bound to the 30S subunit.

Codon, Initiator↗
PubMed2003

The lonepair triloop: a new motif in RNA structure.

The lonepair triloop (LPTL) is an RNA structural motif that contains a single ("lone") base-pair capped by a hairpin loop containing three nucleotides. The two nucleotides immediately outside of this motif (5' and 3' to the lonepair) are not base-paired to one another, restricting the length of this helix to a single base-pair. Four examples of this motif, along with three tentative examples, were initially identified in the 16S and 23S rRNAs with covariation analysis. An evaluation of the recently determined crystal structures of the Thermus thermophilus 30S and Haloarcula marismortui 50S ribosomal subunits revealed the authenticity for all of these proposed interactions and identified 16 more LPTLs in the 5S, 16S and 23S rRNAs. This motif is found in the T loop in the tRNA crystal structures. The lonepairs are positioned, in nearly all examples, immediately 3' to a regular secondary structure helix and are stabilized by coaxial stacking onto this flanking helix. In all but two cases, the nucleotides in the triloop are involved in a tertiary interaction with another section of the rRNA, establishing an overall three-dimensional function for this motif. Of these 24 examples, 14 occur in multi-stem loops, seven in hairpin loops and three in internal loops. While the most common lonepair, U:A, occurs in ten of the 24 LPTLs, the remaining 14 LPTLs contain seven different base-pair types. Only a few of these lonepairs adopt the standard Watson-Crick base-pair conformations, while the majority of the base-pairs have non-standard conformations. While the general three-dimensional conformation is similar for all examples of this motif, characteristic differences lead to several subtypes present in different structural environments. At least one triloop nucleotide in 22 of the 24 LPTLs in the rRNAs and tRNAs forms a tertiary interaction with another part of the RNA. When a LPTL containing the GNR or UYR triloop sequence forms a tertiary interaction with the first (and second) triloop nucleotide, it recruits a fourth nucleotide to mediate stacking and mimic the tetraloop conformation. Approximately half of the LPTL motifs are in close association with proteins. The majority of these LPTLs are positioned at sites in rRNAs that are conserved in the three phylogenetic domains; a few of these occur in regions of the rRNA associated with ribosomal function, including the presumed site of peptidyl transferase activity in the 23S rRNA.

Base Pairing↗
PubMed1998

Archaeon Pyrococcus kodakaraensis KOD1: application and evolution.

Archaea is the third domain which is phylogenetically differentiated from the other two domains, bacteria and eucarya. Hyperthermophile within the archaea domain has evolved most slowly retaining many ancestral features of higher eukaryotes. Pyrococcus kodakaraensis KOD1, which grows at 95 degrees C optimally, is a newly isolated hyperthermophilc archaeon. The KOD1 strain possesses a circular genome, whose size is estimated to be approximately 2,036 kb. KOD1 enzymes involved in the genetic information processing system, such as DNA polymerase, Rec protein, aspartyl tRNA synthetase and molecular chaperonin, share features of eukaryotic enzymes. Rapid and accurate PCR method by KOD1 DNA polymerase and enzyme stabilization system by KOD1 chaperonin are also introduced in this article.

DNA, Archaeal↗
PubMed2002

Highly conserved modified nucleosides influence Mg2+-dependent tRNA folding.

Transfer RNA structure involves complex folding interactions of the TPsiC domain with the D domain. However, the role of the highly conserved nucleoside modifications in the TPsiC domain, rT54, Psi55 and m5C49, in tertiary folding is not understood. To determine whether these modified nucleosides have a role in tRNA folding, the association of variously modified yeast tRNA(Phe) T-half molecules (nucleosides 40-72) with the corresponding unmodified D-half molecule (nucleosides 1-30) was detected and quantified using a native polyacrylamide gel mobility shift assay. Mg2+ was required for formation and maintenance of all complexes. The modified T-half folding interactions with the D-half resulted in K(d)s (rT54 = 6 +/- 2, m5C49 = 11 +/- 2, Psi55 = 14 +/- 5, and rT54,Psi55 = 11 +/- 3 microM) significantly lower than that of the unmodified T-half (40 +/- 10 microM). However, the global folds of the unmodified and modified complexes were comparable to each other and to that of an unmodified yeast tRNA(Phe) and native yeast tRNA(Phe), as determined by lead cleavage patterns at U17 and nucleoside substitutions disrupting the Levitt base pair. Thus, conserved modifications of tRNA's TPsiC domain enhanced the affinity between the two half-molecules without altering the global conformation indicating an enhanced stability to the complex and/or an altered folding pathway.

Base Sequence↗
PubMed1989

On the role of the termination factor RF-2 and the 16S RNA in protein synthesis.

In the translational termination step of protein synthesis the three termination codons UAA, UAG or UGA are recognized by so-called release or termination factors. The release factor RF-1 interacts with UAG and UAA whereas RF-2 is specific for UGA and UAA. Two mechanisms concerning the termination event have been discussed so far: recognition of the termination codon by the protein in a tRNA-like manner or double-strand formation between the codon and the 3' end of the 16S rRNA which is stabilized by the termination factor. Using equilibrium dialysis we show that 40% of the ribosomes can bind UGAA in an RF-2-dependent manner. The stability with the correct combination RF-2-UGA is tenfold higher as compared to the wrong termination codon UAG. We confirm prior findings that the termination factor RF-2 is bound to the A-site of the ribosome. In addition to the ribosomal proteins L2, L10, L7/L12 and L20 of the large subunit and S6 and S18 of the small subunit, the 16S rRNA became labelled when radioactive UGA was crosslinked to the ribosome in the presence of RF-2. Our data support a mechanism of termination in which a double strand between the termination codon and the 3' end of the 16S rRNA is formed as the starting event. The resulting RNA-RNA double strand in turn may be recognized and stabilized by the termination factor.

Binding Sites↗
PubMed2005

Allele-specific conditional destabilization of glutamine repeat mRNAs.

Several late-onset neurological diseases are caused by the inheritance of an expanded CAG repeat coding for polyglutamine. To date there is no effective means of halting the progression of these diseases, and their underlying molecular mechanisms remain a mystery. Strategies designed to specifically reduce the levels of long repeat mRNA might provide an effective therapy for these diseases. An emphasis on allele specificity is necessary to avoid the potential toxicities associated with reduction of expression. The experiments described here are based on the relationship between translation and mRNA stability and the idea that translation of a repeated codon might be extremely sensitive to reductions in levels of cognate aminoacylated tRNA. Consistent with this hypothesis, we have discovered that reduced glutamine concentration destabilizes mRNAs coding for long glutamine repeats while sparing short repeat versions of the same mRNAs. These results suggest therapy might be attained with existing compounds or environmental conditions known to decrease free glutamine levels.

Alleles↗
PubMed1972

Unusual valyl-transfer ribonucleic acid synthetase mutant of Escherichia coli.

Escherichia coli strain NP2907 was isolated as a spontaneous mutant of strain NP29, which possesses a thermolabile valyl-transfer ribonucleic acid (tRNA) synthetase. The valyl-tRNA synthetase of the new mutant, unlike that of its immediate parent, retains enzymatic activity in vitro but differs from the wild-type enzyme in stability and apparent K(m) for adenosine triphosphate. The new mutant locus, valS-102, cotransduces with pyrB at the same frequency as does the parental locus, valS-1. Cultures of strain NP29 cease growth immediately in any medium when shifted from 30 to 40 C. The new mutant grows normally at 30 C, and upon a shift to 40 C growth quickly accelerates exactly as for normal cells. Exponential growth, however, cannot be sustained at 40 C. At a point characteristic for each medium, growth becomes linear with time. This transition occurs almost immediately in rich media and after 1.5 generations in glucose minimal medium. Net synthesis of valyl-tRNA synthetase ceases in the new mutant as soon as the temperature is raised to 40 C, irrespective of the growth medium. We conclude that it is the amount of valyl-tRNA synthetase activity that limits the rate of growth in the linear phase at 40 C. This property of the mutant makes it possible to evaluate the in vivo efficiency of this enzyme at different growth rates and thereby to determine the concentration that is necessary for a given rate of protein synthesis. The results of our measurements indicate that cells of E. coli growing in minimal medium normally possess a functional excess of valyl-tRNA synthetase with respect to protein synthesis and to repression of threonine deaminase.

Adenosine Triphosphate↗
PubMed2005

Roles of conserved amino acid sequence motifs in the SpoU (TrmH) RNA methyltransferase family.

Transfer RNA (Gm18) methyltransferase (TrmH (SpoU)) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to the 2'-OH of guanosine 18 in tRNA. This enzyme is a member of the SpoU family of RNA methyltransferases. Recent computational researches have shown that three amino acid sequence motifs are conserved among the SpoU members. Recently, we determined the crystal structures of the apoand AdoMet bound forms of TrmH (Nureki, O., Watanabe, K., Fukai, S., Ishii, R., Endo, Y., Hori, H., and Yokoyama, S. (2004) Structure 12, 593-602). Furthermore, we clarified the AdoMet binding site and proposed the catalytic mechanism. Since the functions of the conserved amino acid residues in the motifs remain unknown, here we have prepared 17 mutants of TrmH and carried out various biochemical studies, including determination of the kinetic parameters for both AdoMet and tRNA, S-adenosyl-l-homocysteine affinity chromatography, gel mobility shift assay, CD spectroscopy, and analytical gel filtration. Our results show that Asn(35), Arg(41), Glu(124), and Asn(152) are involved in binding tRNA and that the Asn(35) residue is involved in the release of S-adenosyl-l-homocysteine. Several residues of TrmH are important for stability of the enzyme. Taken together, our biochemical studies reinforce the previously proposed catalytic mechanism. We also discuss amino acid substitutions in general within the SPOUT superfamily of methyltransferases.

Amino Acid Motifs↗
PubMed2001

Identification of thermodynamically relevant interactions between EF-Tu and backbone elements of tRNA.

A set of 45 different tRNAs, each containing a single deoxynucleotide substitution covering the upper half of the molecule was used in conjunction with a high-throughput ribonuclease protection assay to investigate the thermodynamic role of 2' hydroxyl groups in stabilizing a complex with elongation factor Tu (EF-Tu) from Thermus thermophilus. Five distinct 2' hydroxyl groups were identified where substitution with a proton resulted in an approximately tenfold decrease in the binding affinity. The same five 2' hydroxyl groups reduced the affinity of the interaction with the nearly identical Thermus aquaticus EF-Tu. Four of these 2' hydroxyl groups were observed to form hydrogen bonds in a co-crystal structure of tRNA(Phe) and T. aquaticus EF-Tu, while the fifth 2' hydroxyl group can be associated with an intramolecular hydrogen bond in the tRNA. However, four additional hydrogen bonds to 2' hydroxyl groups observed in the crystal structure show no thermodynamic effect upon disruption. Some of these discrepancies may be reconciled based on the unbound structures of the protein and RNA.

Alanine↗
PubMed1999

A prototypical cytidylyltransferase: CTP:glycerol-3-phosphate cytidylyltransferase from bacillus subtilis.

BACKGROUND: The formation of critical intermediates in the biosynthesis of lipids and complex carbohydrates is carried out by cytidylyltransferases, which utilize CTP to form activated CDP-alcohols or CMP-acid sugars plus inorganic pyrophosphate. Several cytidylyltransferases are related and constitute a conserved family of enzymes. The eukaryotic members of the family are complex enzymes with multiple regulatory regions or repeated catalytic domains, whereas the bacterial enzyme, CTP:glycerol-3-phosphate cytidylyltransferase (GCT), contains only the catalytic domain. Thus, GCT provides an excellent model for the study of catalysis by the eukaryotic cytidylyltransferases. RESULTS: The crystal structure of GCT from Bacillus subtilis has been determined by multiwavelength anomalous diffraction using a mercury derivative and refined to 2.0 A resolution (R(factor) 0.196; R(free) 0.255). GCT is a homodimer; each monomer comprises an alpha/beta fold with a central 3-2-1-4-5 parallel beta sheet. Additional helices and loops extending from the alpha/beta core form a bowl that binds substrates. CTP, bound at each active site of the homodimer, interacts with the conserved (14)HXGH and (113)RTXGISTT motifs. The dimer interface incorporates part of a third motif, (63)RYVDEVI, and includes hydrophobic residues adjoining the HXGH sequence. CONCLUSIONS: Structure superpositions relate GCT to the catalytic domains from class I aminoacyl-tRNA synthetases, and thus expand the tRNA synthetase family of folds to include the catalytic domains of the family of cytidylyltransferases. GCT and aminoacyl-tRNA synthetases catalyze analogous reactions, bind nucleotides in similar U-shaped conformations, and depend on histidines from analogous HXGH motifs for activity. The structural and other similarities support proposals that GCT, like the synthetases, catalyzes nucleotidyl transfer by stabilizing a pentavalent transition state at the alpha-phosphate of CTP.

Amino Acid Sequence↗
PubMed1996

Overexpression of an mRNA dependent on rare codons inhibits protein synthesis and cell growth.

lambda's int gene contains an unusually high frequency of the rare arginine codons AGA and AGG, as well as dual rare Arg codons at three positions. Related work has demonstrated that Int protein expression depends on the rare AGA tRNA. Strong transcription of the int mRNA with a highly efficient ribosome-binding site leads to inhibition of Int protein synthesis, alteration of the overall pattern of cellular protein synthesis, and cell death. Synthesis or stability of int and ampicillin resistance mRNAs is not affected, although a portion of the untranslated int mRNA appears to be modified in a site-specific fashion. These phenotypes are not due to a toxic effect of the int gene product and can be largely reversed by supplementation of the AGA tRNA in cells which bear plasmids expressing the T4 AGA tRNA gene. This indicates that depletion of the rare Arg tRNA due to ribosome stalling at multiple AGA and AGG codons on the overexpressed int mRNA underlies all of these phenomena. It is hypothesized that int mRNA's effects on protein synthesis and cell viability relate to phenomena involved in lambda phage induction and excision.

Arginine↗
PubMed2006

Evolutionary conservation of a functionally important backbone phosphate group critical for aminoacylation of histidine tRNAs.

All histidine tRNA molecules have an extra nucleotide, G-1, at the 5' end of the acceptor stem. In bacteria, archaea, and eukaryotic organelles, G-1 base pairs with C73, while in eukaryotic cytoplasmic tRNAHis, G-1 is opposite A73. Previous studies of Escherichia coli histidyl-tRNA synthetase (HisRS) have demonstrated the importance of the G-1:C73 base pair to tRNAHis identity. Specifically, the 5'-monophosphate of G-1 and the major groove amine of C73 are recognized by E. coli HisRS; these individual atomic groups each contribute approximately 4 kcal/mol to transition state stabilization. In this study, two chemically synthesized 24-nucleotide RNA microhelices, each of which recapitulates the acceptor stem of either E. coli or Saccharomyces cervisiae tRNAHis, were used to facilitate an atomic group "mutagenesis" study of the -1:73 base pair recognition by S. cerevisiae HisRS. Compared with E. coli HisRS, microhelixHis is a much poorer substrate relative to full-length tRNAHis for the yeast enzyme. However, the data presented here suggest that, similar to the E. coli system, the 5' monophosphate of yeast tRNA(His) is critical for aminoacylation by yeast HisRS and contributes approximately 3 kcal/mol to transition state stability. The primary role of the unique -1:73 base pair of yeast tRNAHis appears to be to properly position the critical 5' monophosphate for interaction with the yeast enzyme. Our data also suggest that the eukaryotic HisRS/tRNAHis interaction has coevolved to rely less on specific major groove interactions with base atomic groups than the bacterial system.

Acylation↗
PubMed2007

Importance of A-loop complementarity with tRNAHis anticodon for continued selection of tRNAHis as the HIV reverse transcription primer.

BACKGROUND: Human immunodeficiency virus (HIV-1) preferentially selects tRNALys,3 as the primer for reverse transcription. HIV-1 can be forced to select alternative tRNAs through mutation in the primer-binding site (PBS) and a region upstream of the PBS designated as the A-loop. Alteration of the PBS and A-loop to be complementary to the 3' terminal nucleotides and anticodon of tRNAHis results in HIV-1 that can stably utilize this tRNA for replication. RESULTS: In the current study, we have investigated the effect that mutations within the A-loop have on the stability of HIV-1 with a PBS complementary to tRNAHis. For these studies, we have altered the A-loop to be complementary to tRNAMet, tRNAGln, tRNAIle, tRNAThr and tRNASer. All substitutions of the A-loops with the PBS complementary to tRNAHis resulted in a reduction of infectious virus obtained following transfection of proviral genomes in the 293T cells. Virus replication in SupT1 cells was also impaired as a result of the alteration of the A-loop. Viruses with the A-loop complementary to tRNALys,3 and tRNASer reverted to utilize tRNALys,3 following in vitro replication. In contrast, viruses with the A-loop complementary to the other tRNAs remained stable and continued to use tRNAHis. RNA modeling of the stem-loop structure revealed that nucleotides were displayed on the loop region that could potentially interact with the anticodon of tRNAHis. To further explore the effects of the A-loop mutations on virus replication, the A-loops complementary to tRNASer or tRNAHis were cloned into the wild type genome with the PBS complementary to tRNALys,3. Transfection of proviral genomes which contained the wild type PBS and A-loops complementary to tRNASer or tRNAHis into 293 T cells did not impact on the production of viruses as measured by p24 antigen ELISA. However, viruses with the A-loop complementary to tRNAHis had greatly reduced infectivity and replicated poorly in SupT1 compared to the wild type or viruses with the A-loop complementary to tRNASer. CONCLUSION: These studies demonstrate that complementarity of A-loop region with the anticodon of tRNAHis has a pronounced effect on the capacity of HIV-1 to utilize tRNAHis as the primer for reverse transcription. Complementarity between A-loop and anticodon of the tRNA then is important for the selection of the tRNA primer used for reverse transcription.

Anticodon↗
PubMed2001

Translation initiation factor IF3: two domains, five functions, one mechanism?

Initiation factor IF3 contains two domains separated by a flexible linker. While the isolated N-domain displayed neither affinity for ribosomes nor a detectable function, the isolated C-domain, added in amounts compensating for its reduced affinity for 30S subunits, performed all activities of intact IF3, namely: (i) dissociation of 70S ribosomes; (ii) shift of 30S-bound mRNA from 'stand-by' to 'P-decoding' site; (iii) dissociation of 30S-poly(U)-NacPhe-tRNA pseudo- initiation complexes; (iv) dissociation of fMet-tRNA from initiation complexes containing mRNA with the non-canonical initiation triplet AUU (AUUmRNA); (v) stimulation of mRNA translation regardless of its start codon and inhibition of AUUmRNA translation at high IF3C/ribosome ratios. These results indicate that while IF3 performs all its functions through a C-domain-30S interaction, the N-domain function is to provide additional binding energy so that its fluctuating interaction with the 30S subunit can modulate the thermodynamic stability of the 30S-IF3 complex and IF3 recycling. The localization of IF3C far away from the decoding site and anticodon stem-loop of P-site-bound tRNA indicates that the IF3 fidelity function does not entail its direct contact with these structures.

Escherichia coli↗
PubMed2003

On the structural and functional importance of the highly conserved Glu56 of Thermus thermophilus L4 ribosomal protein.

The structural and functional importance of the highly conserved amino acid residue glutamic acid 56 (Glu56) of the ribosomal protein L4 from Thermus thermophilus (TthL4) has been investigated by replacing this residue by alanine or glutamine, and by incorporating the resulted mutants into Escherichia coli ribosomes. The catalytic properties of peptidyltransferase estimated for the mutants as well as for the wild-type TthL4 by the puromycin reaction, were quite different. The binding of tRNA to the P and A-site was affected. In addition, replacement of the native L4 protein by wild-type TthL4 or by TthL4-Ala56 mutant resulted in reduced capability of 50S subunits for association with 30S subunits. In contrast, neither the assembly of the 50S subunits nor the fixation of the tRNA 3'-end at the P or A-site was affected. These results are used to discuss critically the hypothesis that the delta-carboxyl group of the highly conserved Glu56 is essential for stabilizing a flexible loop of L4, which extended into the ribosome interior region, influences the mechanism of peptide bond formation. Mutations concerning the semi-conserved glycine 55 (Gly55) were investigated. Replacement of Gly55 by serine did not affect the measured functions. In contrast, replacement of Gly55 by alanine resulted in enhanced peptidyltransferase activity and increased tRNA affinity for the P and A-sites, indicating a possible implication of this amino acid in the local loop conformation of TthL4.

Alanine↗
PubMed1994

A tyrosyl-tRNA synthetase can function similarly to an RNA structure in the Tetrahymena ribozyme.

Group I introns are highly structured RNAs which catalyse their own splicing by guanosine-initiated transesterification reactions. Their catalytic core is generally stabilized by RNA-RNA interactions within the core and with peripheral RNA structures. Additionally, some group I introns require proteins for efficient splicing in vivo. The Neurospora CYT-18 protein, the mitochondrial tyrosyl-transfer RNA synthetase (mt TyrRS), promotes splicing of the Neurospora mitochondrial large ribosomal RNA (LSU) and other group I introns by stabilizing the catalytically active structure of the intron core. We report here that CYT-18 functions similarly to a peripheral RNA structure, P5abc, that stabilizes the catalytic core of the Tetrahymena LSU intron. The CYT-18 protein and P5abc RNA bind to overlapping sites in the intron core, inducing similar conformational changes correlated with splicing activity. Our results show that a protein can play the role of an RNA structure in a catalytic RNA, a substitution postulated for the evolution of nuclear pre-messenger RNA introns from self-splicing introns.

Animals↗
PubMed1991

Lysine 335, part of the KMSKS signature sequence, plays a crucial role in the amino acid activation catalysed by the methionyl-tRNA synthetase from Escherichia coli.

The KMSKS pattern, conserved among several aminoacyl-tRNA synthetase sequences, was first recognized in the Escherichia coli methionyl-tRNA synthetase through affinity labelling with an oxidized reactive derivative of tRNA(Met)f. Upon complex formation, two lysine residues of the methionyl-tRNA synthetase (Lys61 and 335, the latter being part of the KMSKS sequence) could be crosslinked by the 3'-acceptor end of the oxidized tRNA. Identification of an equivalent reactive lysine residue at the active centre of tyrosyl-tRNA synthetase designated the KMSKS sequence as a putative component of the active site of methionyl-tRNA synthetase. To probe the functional role of the labelled lysine residue within the KMSKS pattern, two variants of methionyl-tRNA synthetase containing a glutamine residue at either position 61 or 335 were constructed by using site-directed mutagenesis. Substitution of Lys61 slightly affected the enzyme activity. In contrast, the enzyme activities were very sensitive to the substitution of Lys335 by Gln. Pre-steady-state analysis of methionyladenylate synthesis demonstrated that this substitution rendered the enzyme unable to stabilize the transition state complex in the methionine activation reaction. A similar effect was obtained upon substituting Lys335 by an alanine instead of a glutamine residue, thereby excluding an effect specific for the glutamine side-chain. Furthermore, the importance of the basic character of Lys335 was investigated by studying mutants with a glutamate or an arginine residue at this position. It is concluded that the N-6-amino group of Lys335 plays a crucial role in the activation of methionine, mainly by stabilizing the transient complex on the way to methionyladenylate, through interaction with the pyrophosphate moiety of bound ATP-Mg2+. We propose, therefore, that the KMSKS pattern in the structure of an aminoacyl-tRNA synthetase sequence represents a signature sequence characteristic of both the pyrophosphate subsite and the catalytic centre.

Adenosine Triphosphate↗