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At least 469 records · Page 26Linked to original sources
PubMed1990

Overproduction of tyrosyl-tRNA synthetase is toxic to Escherichia coli: a genetic analysis.

The tyrS genes from Escherichia coli and Bacillus stearothermophilus were toxic to E. coli when they were carried by plasmids with very high copy numbers (pEMBL8 and pEMBL9). We quantified this effect by comparing the efficiencies of plating of E. coli derivatives harboring recombinant plasmids in various experimental conditions. The toxicity was apparent at both 30 and 37 degrees C. It increased with the growth temperature, the strength of the tyrS promoter, and the copy number of the plasmidic vector. Two- to threefold enhancement of tyrS expression raised the toxicity 300-fold. Point mutations in tyrS that prevent interaction between its product, tyrosyl-tRNA synthetase, and tRNA(Tyr) but do not alter the rate of formation of tyrosyl-adenylate abolished the toxicity. Thus, the toxic effect was due to high cellular levels of synthetase activity. At 30 degrees C, the cellular concentration of tyrosyl-tRNA synthetase reached 55% of that of soluble proteins and led to decreased beta-galactosidase stability. We discuss possible causes of this toxic effect and describe its applications to the study of the recognition and interaction between the synthetase and tRNA(Tyr).

Amino Acyl-tRNA Synthetases↗
PubMed2005

Structural transitions induced by the interaction between tRNA(Gly) and the Bacillus subtilis glyQS T box leader RNA.

The T box system regulates expression of amino acid-related genes in Gram-positive bacteria through premature termination of transcription. Synthesis of the full-length mRNA requires stabilization of an antiterminator element in the 5' untranslated leader RNA by the cognate uncharged tRNA. tRNA(Gly)-dependent antitermination of the Bacillus subtilis glyQS gene (encoding glycyl-tRNA synthetase) can be reproduced in a purified in vitro transcription system, indicating that the nascent transcript is sufficient for interaction with the tRNA. Genetic analyses previously demonstrated base pairing of a single codon in the leader RNA with the tRNA anticodon, and between the antiterminator and the tRNA acceptor end. In this study, we established conditions for specific binding of tRNA(Gly) to glyQS leader RNA generated by phage T7 RNA polymerase. Structural mapping studies revealed tRNA(Gly)-induced protection in the glyQS leader RNA at the two known sites of interaction with the tRNA, as well as at other regions between these sites. The proposed tRNA-dependent structural switch between the competing terminator and antiterminator forms of the leader RNA was demonstrated directly. Changes in tRNA(Gly) upon binding to glyQS leader RNA were detected in the anticodon loop, consistent with pairing with the specifier sequence, and in the highly conserved G19 in the D-loop, similar to effects induced by codon-anticodon interaction in the ribosome. This study provides biochemical evidence for direct interaction of tRNA(Gly) with full-length in vitro transcribed glyQS leader RNA, and an initial view of structural modulations of both RNA partners within the complex.

5' Untranslated Regions↗
PubMed2006

Structure of eEF3 and the mechanism of transfer RNA release from the E-site.

Elongation factor eEF3 is an ATPase that, in addition to the two canonical factors eEF1A and eEF2, serves an essential function in the translation cycle of fungi. eEF3 is required for the binding of the aminoacyl-tRNA-eEF1A-GTP ternary complex to the ribosomal A-site and has been suggested to facilitate the clearance of deacyl-tRNA from the E-site. Here we present the crystal structure of Saccharomyces cerevisiae eEF3, showing that it consists of an amino-terminal HEAT repeat domain, followed by a four-helix bundle and two ABC-type ATPase domains, with a chromodomain inserted in ABC2. Moreover, we present the cryo-electron microscopy structure of the ATP-bound form of eEF3 in complex with the post-translocational-state 80S ribosome from yeast. eEF3 uses an entirely new factor binding site near the ribosomal E-site, with the chromodomain likely to stabilize the ribosomal L1 stalk in an open conformation, thus allowing tRNA release.

Adenosine Diphosphate↗
PubMed2001

A role for initiation codon context in chloroplast translation.

To study the role of initiation codon context in chloroplast protein synthesis, we mutated the three nucleotides immediately upstream of the initiation codon (the -1 triplet) of two chloroplast genes in the alga Chlamydomonas reinhardtii. In prokaryotes, the -1 triplet has been proposed to base pair with either the 530 loop of 16S rRNA or the extended anticodon of fMet-tRNA. We found that in vivo, none of the chloroplast mutations affected mRNA stability. However, certain mutations did cause a temperature-sensitive decrease in translation and a more dramatic decrease at room temperature when combined with an AUU initiation codon. These mutations disrupt the proposed extended base pairing interaction with the fMet-tRNA anticodon loop, suggesting that this interaction may be important in vivo. Mutations that would still permit base pairing with the 530 loop of the 16S rRNA also had a negative effect on translation, suggesting that this interaction does not occur in vivo. Extended base pairing surrounding the initiation codon may be part of a mechanism to compensate for the lack of a classic Shine-Dalgarno rRNA interaction in the translation of some chloroplast mRNAs.

Animals↗
PubMed2004

HIV type 1 that select tRNA(His) or tRNA(Lys1,2) as primers for reverse transcription exhibit different infectivities in peripheral blood mononuclear cells.

The replication in human peripheral blood mononuclear cells (PBMC) of unique HIV-1 that select tRNA(His) or tRNA(Lys1,2) for reverse transcription was compared to the wild-type virus that uses tRNA(Lys,3). HIV-1 with only the primer-binding site (PBS) changed to be complementary to these alternative tRNAs initially replicated more slowly than the wild-type virus in PBMC, although all viruses eventually reached equivalent growth as measured by p24 antigen. Viruses with only a PBS complementary to the 3' terminal 18 nucleotides of tRNA(His) or tRNA(Lys1,2) reverted to use tRNA(Lys3). HIV-1 with mutations in the U5-PBS to allow selection of tRNA(His) and tRNA(Lys1,2) following long-term growth in SupT1 cells were also evaluated for growth and PBS stability following replication in PBMC. Although both viruses initially grew slower than wild type, they maintained a PBS complementary to the starting tRNA and did not revert to the wild-type PBS after long-term culture in PBMC. Analysis of the U5-PBS regions following long-term culture in PBMC also revealed few changes from the starting sequences. The virus that stably used tRNA(His) was less infectious than the wild type. In contrast, the virus that stably used tRNA(Lys1,2) evolved to be as infectious as wild-type virus following extended culture in PBMC. The results of these studies highlight the impact of the host cell on the tRNA primer selection process and subsequent infectivity of HIV-1.

Base Sequence↗
PubMed1998

Vibrational dynamics of transfer RNAs: comparison of the free and synthetase-bound forms.

The vibrational dynamics of transfer RNAs, both free, and complexed with the cognate synthetase, are analyzed using a model (Gaussian network model) which recently proved to satisfactorily describe the collective motions of folded proteins. The approach is similar to a normal mode analysis, with the major simplification that no residue specificity is taken into consideration, which permits us (i) to cast the problem into an analytical form applicable to biomolecular systems including about 10(3 )residues, and (ii) to acquire information on the essential dynamics of such large systems within computational times at least two orders of magnitude shorter than conventional simulations. On a local scale, the fluctuations calculated for yeast tRNAPhe and tRNAAsp in the free state, and for tRNAGln complexed with glutaminyl-tRNA synthetase (GlnRS) are in good agreement with the corresponding crystallographic B factors. On a global scale, a hinge-bending region comprising nucleotides U8 to C12 in the D arm, G20 to G22 in the D loop, and m7G46 to C48 in the variable loop (for tRNAPhe), is identified in the free tRNA, conforming with previous observations. The two regions subject to the largest amplitude anticorrelated fluctuations in the free form, i.e. the anticodon region and the acceptor arm are, at the same time, the regions that experience the most severe suppression in their flexibilities upon binding to synthetase, suggesting that their sampling of the conformational space facilitates their recognition by the synthetase. Likewise, examination of the global mode of motion of GlnRS in the complex indicates that residues 40 to 45, 260 to 270, 306 to 314, 320 to 327 and 478 to 485, all of which cluster near the ATP binding site, form a hinge-bending region controlling the cooperative motion, and thereby the catalytic function, of the enzyme. The distal beta-barrel and the tRNA acceptor binding domain, on the other hand, are distinguished by their high mobilities in the global modes of motion, a feature typical of recognition sites, also observed for other proteins. Most of the conserved bases and residues of tRNA and GlnRS are severely constrained in the global motions of the molecules, suggesting their having a role in stabilizing and modulating the global motion.

Amino Acyl-tRNA Synthetases↗
PubMed1999

Structural and functional roles of the N1- and N3-protons of psi at tRNA's position 39.

Pseudouridine at position 39 (Psi(39)) of tRNA's anticodon stem and loop domain (ASL) is highly conserved. To determine the physicochemical contributions of Psi(39)to the ASL and to relate these properties to tRNA function in translation, we synthesized the unmodified yeast tRNA(Phe)ASL and ASLs with various derivatives of U(39)and Psi(39). Psi(39)increased the thermal stability of the ASL (Delta T (m)= 1.3 +/- 0.5 degrees C), but did not significantly affect ribosomal binding ( K (d)= 229 +/- 29 nM) compared to that of the unmodified ASL (K (d)= 197 +/- 58 nM). The ASL-Psi(39)P-site fingerprint on the 30S ribosomal subunit was similar to that of the unmodified ASL. The stability, ribosome binding and fingerprint of the ASL with m(1)Psi(39)were comparable to that of the ASL with Psi(39). Thus, the contribution of Psi(39)to ASL stability is not related to N1-H hydrogen bonding, but probably is due to the nucleoside's ability to improve base stacking compared to U. In contrast, substitutions of m(3)Psi(39), the isosteric m(3)U(39)and m(1)m(3)Psi(39)destabilized the ASL by disrupting the A(31)-U(39)base pair in the stem, as confirmed by NMR. N3-methylations of both U and Psi dramatically decreased ribosomal binding ( K (d)= 1060 +/- 189 to 1283 +/- 258 nM). Thus, canonical base pairing of Psi(39)to A(31)through N3-H is important to structure, stability and ribosome binding, whereas the increased stability and the N1-proton afforded by modification of U(39)to Psi(39)may have biological roles other than tRNA's binding to the ribosomal P-site.

Anticodon↗
PubMed1994

Effect of the nucleotide-37 on the interaction of tRNA(Phe) with the P site of Escherichia coli ribosomes.

The method of anticodon loop replacement has been used to make derivatives of yeast tRNA(Phe) with the substitution at position 37 (tRNA(Phe)GAAA) and at the anticodon(tRNA(Phe)GCAG). A quantitative study of the interaction of various types of deacylated yeast tRNA(Phe) (tRNA(Phe)+Y, tRNA(Phe)GAAA, tRNA(Phe)-Y) with the P site of the [70S ribosome*poly(U)]-complex was carried out at different Mg2+ concentrations and temperatures. The presence and nature of the nucleotide situated at the 3'-end of the anticodon are essential for such interaction in E coli ribosomes. Replacement of the Y base with the unmodified adenosine decreases the interaction enthalpy from 39 kcal/mol to 24 kcal/mol, whereas its removal reduces the interaction enthalpy to 16 kcal/mol. Replacement of the second anticodon nucleotide, adenosine, with cytosine further reduces the enthalpy to 6 kcal/mol, which is typical of tRNA-P site interaction in the absence of poly(U). In the absence of poly(U) the affinity of tRNA(PheY) for the P site of the 70S ribosome is five times lower than the affinity of tRNA(Phe+Y) or tRNA(Phe)GCAG. Thus, in the ribosome the modified nucleotide stabilizes the codon-anticodon interaction through its stacking interaction with the codon-anticodon base stack. In addition, this decreases the free energy of binding as a result of the interaction of the modified nucleotide itself with the hydrophobic center of the P site.

Acylation↗
PubMed2006

Optimization of a mouse recombinant antibody fragment for efficient production from Escherichia coli.

A mutagenized mouse recombinant antibody fragment (rFab) that recognized HIV capsid protein was isolated from Escherichia coli at a level of 12 mg per liter of culture using standard shake flask methods. This is one of the highest yields of a modified antibody fragment obtained using non-fermentor-based methods. Recombinant Fab was isolated directly from the culture medium, which lacked complex materials such as tryptone and yeast extract. Fab isolated from the periplasm was not as homogeneous as that isolated directly from the culture medium. Optimization of the culture medium using recently developed media, the use of E. coli cell lines that contained rare tRNA codons, and mutagenesis of the Fab to improve the stability of the Fab were important factors in producing high-levels of the Fab. An isolation protocol easily adaptable to automation using a thiophilic-sepharose column followed by metal-chelate chromatography and the introduction of a non-traditional metal binding site for metal-chelate purification that bypasses the conventional hexahistidine tag cleavage step (to prevent the purification tag from interfering with crystallization) are additional features of this approach to produce a highly homogenous preparation of rFab. The resulting rFab binds to its antigen, p24, equivalent in character to the monoclonal from which the rFab was originally derived.

Amino Acid Sequence↗
PubMed1982

Mechanistic studies of ribonucleic acid renaturation by a helix-destabilizing protein.

The ability of a nucleic acid helix-destabilizing protein from calf thymus, UP1, to facilitate renaturation of yeast tRNALeu3 and Escherichia coli 5S RNA is shown to be a consequence of the protein's ability to bind stoichiometrically to single-stranded polynucleotide regions. A comparison of the inhibitory effect of different homopolymers on UP1-induced renaturation of tRNALeu3 does not indicate significant base specificity in UP1 binding, and a 3'-5' ribose phosphate polymer devoid of heterocyclic bases inhibits as well as the homopolynucleotides. These inhibition studies also show that UP1 requires polynucleotide segments of at least three phosphate residues to bind. Mg2+ (which is required for the stabilization of native tRNALeu3) dissociates complexes of UP1 with inactive tRNA, and since the RNAs in those complexes lack a substantial amount of secondary structure, it can upon dissociation readily refold into the native structure. A semiquantitative treatment of UP1-RNA interaction is developed that suggests that only a small number (approximately six) of protein molecules are bound to tRNALeu3 in the complex while analysis of the inhibition studies suggests that these UP1 molecules are not bound in a highly cooperative manner.

Animals↗
PubMed1985

Self-catalyzed cyclization of the intervening sequence RNA of Tetrahymena: inhibition by intercalating dyes.

The intervening sequence (IVS) excised from the pre-rRNA of Tetrahymena undergoes a self-catalyzed cleavage-ligation reaction to form a covalently closed circular RNA. This cyclization reaction is kinetically inhibited by ethidium bromide (50% inhibition at 22 +/- 14 microM, greater than 99% inhibition at 53 +/- 16 microM for a 20 minute reaction). The dye does not alter the sites of the cyclization reaction, but it does increase the relative amount of reaction at a minor site 19 nucleotides from the 5' end of the IVS. The reversibility of the inhibition and the relative inhibitory strength of acridine orange, ethidium and proflavine suggest that inhibition is due to intercalation of the dye in functionally important secondary or tertiary structures of the IVS. The concentration of dye required to inhibit cyclization is much higher than expected from the known binding constants of such dyes to tRNA. At high Mg2+ to Na+ ratios, conditions which should stabilize RNA structure, a subpopulation of the IVS RNA molecules is resistant to ethidium inhibition, even at 200 microM ethidium. These data are interpreted as reflecting two conformational isomers of the IVS that differ in their reactivity and in their sensitivity to dye binding.

Acridine Orange↗
PubMed2003

Effect of G-1 on histidine tRNA microhelix conformation.

Histidine tRNAs (tRNA(His)) are unique in that they possess an extra 5'-base (G-1) not found in other tRNAs. Deletion of G-1 results in at least a 250-fold reduction in the rate of histidine charging in vitro. To better understand the role of the G-1 nucleotide in defining the structure of tRNA(His), and to correlate structure with cognate amino acid charging, NMR and molecular dynamics (MD) studies were performed on the wild-type and a DeltaG-1 mutant Escherichia coli histidine tRNA acceptor stem microhelix. Using NMR-derived distance restraints, global structural characteristics are described and interpreted to rationalize experimental observations with respect to aminoacylation activity. The quality of the NMR-derived solution conformations of the wild-type and DeltaG-1 histidine microhelices (micro helix(His)) is assessed using a variety of MD-based computational protocols. Most of the duplex regions of the acceptor stem and the UUCG tetraloop are well defined and effectively superimposable for the wild-type and DeltaG-1 mutant microhelix(His). Differences, however, are observed at the end of the helix and in the single-stranded CCCA-3' tail. The wild-type microhelix(His) structure is more well defined than the mutant and folds into a 'stacked fold-back' conformation. In contrast, we observe fraying of the first two base pairs and looping back of the single-stranded region in the DeltaG-1 mutant resulting in a much less well defined conformation. Thus the role of the extra G-1 base of the unique G-1:C73 base pair in tRNA(His) may be to prevent end-fraying and stabilize the stacked fold-back conformation of the CCCA-3' region.

Base Sequence↗
PubMed1982

Differential synthesis rates of tRNA species in the silk gland of Bombyx mori are required to promote tRNA adaptation to silk messages.

In studying the mechanism of tRNA adaptation to silk fibroin and sericin mRNA translation, we investigated the accumulation rates of tRNA species rapidly labeled in vivo in the posterior and middle silk glands of the silkworm Bombyx mori during the last larval instar. We found that the rates of synthesis of total and individual tRNA species labeled with [3H]uridine during the growth and the secretion phases and separated by electrophoresis on polyacrylamide gels, are specific for each tRNA species and each tissue. We noted continuous variations of the tRNA levels designed to reflect changes in the population of mRNAs. The tRNA adaptation to silk mRNAs is completed roughly at the end of the growth phase and at the beginning of the secretion phase (day four of the Vth instar). Its stabilization during the secretion phase occurs by a reversal of the relative rates of predominant tRNA species, mainly tRNAGly. The sum total of the synthetic rates for each tRNA species corroborates their known adaptive distribution during the secretion phase of silk proteins. In addition, when coupled to the similarity of their turnover rates, observed previously, the data support a transcriptional control of tRNA genes.

Animals↗
PubMed2001

The La protein.

Ubiquitous in eukaryotic cells, the La protein associates with the 3' termini of many newly synthesized small RNAs. RNAs bound by the La protein include all nascent transcripts made by RNA polymerase III as well as certain small RNAs synthesized by other RNA polymerases. Recent genetic and biochemical analyses have revealed that binding by the La protein protects the 3' ends of these RNAs from exonucleases. This La-mediated stabilization is required for the normal pathway of pre-tRNA maturation, facilitates assembly of small RNAs into functional RNA-protein complexes, and contributes to nuclear retention of certain small RNAs. Studies of mutant La proteins have given some insights into how the La protein specifically recognizes its RNA targets. However, many questions remain regarding the molecular mechanisms by which La protein binding influences multiple steps in small RNA biogenesis. This review focuses on the roles of the La protein in small RNA biogenesis and also discusses data that implicate the La protein in the translation of specific mRNAs.

Adenosine Triphosphatases↗
PubMed1983

Translational stability of plant viral RNAs microinjected into living cells. Influence of a 3'-poly(A) segment.

Three different alternative structural features have been shown to be present at the 3' terminus of plant viral RNAs: (a) a poly(A) track, (b) a tRNA-like structure, (c) no special structural or sequence characteristic. We have compared the translational stability after injection into frog oocytes of a representative of each type: (a) the small genomic RNA (M-RNA) of cowpea mosaic virus (CPMV), (b) the subgenomic mRNA for coat protein (RNA 4) of brome mosaic virus (BMV), (c) the subgenomic mRNA for coat protein (RNA 4) of alfalfa mosaic virus (AIMV). It has been shown that CPMV M-RNA exhibits the highest translational stability. However, the stability of AIMV RNA 4 is remarkably high and moreover significantly higher than that of BMV RNA 4. We demonstrate that, for all three viral RNA species considered, the presence of a poly(A) segment at the 3' end of the molecules improves the translational stability. From a comparative investigation in which AIMV RNA 4 was also injected into HeLa cells, it is concluded that the stability of a given non-adenylylated mRNA depends on the nature of the cytoplastic environment.

Animals↗
PubMed2006

A counterintuitive Mg2+-dependent and modification-assisted functional folding of mitochondrial tRNAs.

Mitochondrial tRNAs (mtRNAs) often lack domains and posttranscriptional modifications that are found in cytoplasmic tRNAs. These structural and chemical elements normally stabilize the folding of cytoplasmic tRNAs into canonical structures that are competent for aminoacylation and translation. For example, the dihydrouridine (D) stem and loop domain is involved in the tertiary structure of cytoplasmic tRNAs through hydrogen bonds and a Mg2+ bridge to the ribothymidine (T) stem and loop domain. These interactions are often absent in mtRNA because the D-domain is truncated or missing. Using gel mobility shift analyses, UV, circular dichroism and NMR spectroscopies and aminoacylation assays, we have investigated the functional folding interactions of chemically synthesized and site-specifically modified mitochondrial and cytoplasmic tRNAs. We found that Mg2+ is critical for folding of the truncated D-domain of bovine mtRNAMet with the tRNA's T-domain. Contrary to the expectation that Mg2+ stabilizes RNA folding, the mtRNAMet D-domain structure was unfolded and relaxed, rather than stabilized in the presence of Mg2+. Because the D-domain is transcribed prior to the T-domain, we conclude that Mg2+ prevents misfolding of the 5'-half of bovine mtRNAMet facilitating its correct interaction with the T-domain. The interaction of the mtRNAMet D-domain with the T-domain was enhanced by a pseudouridine located in either the D or T-domains compared to that of the unmodified RNAs (Kd=25.3, 24.6 and 44.4 microM, respectively). Mg2+ also affected the folding interaction of a yeast mtRNALeu1, but had minimal effect on the folding of an Escherichia coli cytoplasmic tRNALeu. The D-domain modification, dihydrouridine, facilitated mtRNALeu folding. These data indicate that conserved modifications assist and stabilize the formation of the functional mtRNA tertiary structure.

Aminoacylation↗