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PubMed1984

Missense and nonsense suppressors derived from a glycine tRNA by nucleotide insertion and deletion in vivo.

Beginning with a missense suppressor tRNA and a nonsense suppressor tRNA, both in Escherichia coli and each containing an extra nucleotide in the anticodon loop, we generated new suppressors in vivo by spontaneous deletion of specific nucleotides from the anticodon loop. In one experiment, the new suppressor was generated by a double mutational event, base substitution and nucleotide deletion. A novel ochre suppressor is also described. It is very efficient in nonsense suppression but has no ms2i6 modification of the A residue on the 3' side of the anticodon. The results have important implications for tRNA structure-function relationships, tRNA recognition by tRNA-modifying enzymes, mechanisms of deletion mutation, and tRNA evolution.

Anticodon↗
PubMed1998

5' processing of tRNA precursors can Be modulated by the human La antigen phosphoprotein.

Eukaryotic precursor (pre)-tRNAs are processed at both ends prior to maturation. Pre-tRNAs and other nascent transcripts synthesized by RNA polymerase III are bound at their 3' ends at the sequence motif UUUOH [3' oligo(U)] by the La antigen, a conserved phosphoprotein whose role in RNA processing has been associated previously with 3'-end maturation only. We show that in addition to its role in tRNA 3'-end maturation, human La protein can also modulate 5' processing of pre-tRNAs. Both the La antigen's N-terminal RNA-binding domain and its C-terminal basic region are required for attenuation of pre-tRNA 5' processing. RNA binding and nuclease protection assays with a variety of pre-tRNA substrates and mutant La proteins indicate that 5' protection is a highly selective activity of La. This activity is dependent on 3' oligo(U) in the pre-tRNA for interaction with the N-terminal RNA binding domain of La and interaction of the C-terminal basic region of La with the 5' triphosphate end of nascent pre-tRNA. Phosphorylation of La is known to occur on serine 366, adjacent to the C-terminal basic region. We show that this modification interferes with the La antigen's ability to protect pre-tRNAiMet from 5' processing either by HeLa extract or purified RNase P but that it does not affect interaction with the 3' end of pre-tRNA. These findings provide the first evidence to indicate that tRNA 5'-end maturation may be regulated in eukaryotes. Implications of triphosphate recognition is discussed as is a role for La phosphoprotein in controlling transcriptional and posttranscriptional events in the biogenesis of polymerase III transcripts.

Adenosine Triphosphatases↗
PubMed1998

Recognition of the 5' leader and the acceptor stem of a pre-tRNA substrate by the ribozyme from Bacillus subtilis RNase P.

The catalysis by the ribozyme from bacterial RNase P involves specific interactions with the structure of the tRNA substrate. Recognition of the T stem-loop by this ribozyme occurs in a groove-like structure dictated by the tertiary folding of tRNA [Loria, A., and Pan, T. (1997) Biochemistry 36, 6317]. Effects of 2'-OH --> 2'-H modifications within the acceptor stem and the 5' leader on substrate binding and catalysis are determined using a tRNAPhe substrate that is significantly cleaved at more than one site. In all but one case, the 2'-deoxy substitution has little effect on binding for cleavage at the correct and incorrect sites. Substitution of the 2'-OH group at the correct site, however, decreases the cleavage chemistry by more than 3.4 kcal/mol for cleavage at both the correct and incorrect sites. Substitutions of the 2'-OH groups at the incorrect sites have no effect for cleavage at the incorrect and correct sites. Truncation of the 5' leader results in differential effects on cleavage at different sites. These observations lead to a model in which cleavage at the correct and incorrect sites involves formation of different ribozyme-substrate complexes depending on binding of specific nucleotides in the 5' leader. Binding of the T stem-loop of tRNA and the 2'-OH group at the correct cleavage site is common for all ES complexes. An A/U-rich 5' leader significantly promotes formation of the ES complex and accelerates the cleavage chemistry over those of a C/G-rich 5' leader, but only moderately enhances cleavage at the correct site over cleavage at the incorrect sites. Since cleavage at different sites requires formation of different ES complexes, cleavage site selection can occur at the level of the ES complex and at the chemical step.

Bacillus subtilis↗
PubMedDate not supplied

[mRNA-binding site of ribosomes at different stages of translation. II. Affinity modification of Escherichia coli ribosomes bya benzylidene derivative of AUGU6 in pre- and post-translation complexes].

Affinity labeling of E. coli ribosomes with the 2',3'-O-[4-(N-2-chloroethyl)-N-methyl-amino]benzylidene derivative of AUGU6 (AUGU6-[14C]CHRCl) was studied within the pretranslocational complex ribosome.AUGU6[14C]CHRCl.tRNA(fMet)(P-site).fMetPhe-tR NA(Phe)(A-site) and posttranslocational complex ribosome.AUGU6[14C]CHRCl.fMetPhe-tRNA(Phe)(P-site). Both 30S and 50S subunits were labeled within these complexes, but the extent of 30S subunit modification was 6-8-fold higher than those for 50S subunit. Ribosomal proteins of both subunits were found to be labeled preferentially. Proteins S1, S5, S11, L1 were identified to be crosslinked with AUGU6[14C]CHRCl within the pretranslocational complex and S7--within the posttranslocational complex from the data of two-dimensional electrophoresis in the polyacrylamide gel.

Affinity Labels↗
PubMed1999

Applicability of urea in the thermodynamic analysis of secondary and tertiary RNA folding.

The equilibrium folding of a series of self-complementary RNA duplexes and the unmodified yeast tRNA(Phe) is studied as a function of urea and Mg(2+) concentration with optical spectroscopies and chemical modification under isothermal conditions. Via application of standard methodologies from protein folding, the folding free energy and its dependence on urea concentration, the m value, are determined. The free energies of the RNA duplexes obtained from the urea titrations are in good agreement with those calculated from thermal melting studies [Freier, S. I., et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9373]. The m value correlates with the length of the RNA duplex and is not sensitive to ionic conditions and temperature. The folding of the unmodified yeast tRNA(Phe) can be described by two Mg(2+)-dependent transitions, the second of which corresponds to the formation of the native tertiary structure as confirmed by hydroxyl radical protection and partial nuclease digestion. Both transitions are sensitive to urea and have m values of 0.94 and 1.70 kcal mol(-)(1) M(-)(1), respectively. Although the precise chemical basis of urea denaturation of RNA is uncertain, the m values for the duplexes and tRNA(Phe) are proportional to the amount of the surface area buried in the folding transition. This proportionality, 0.099 cal mol(-)(1) M(-)(1) A(-)(2), is very similar to that observed for proteins, 0.11 cal mol(-)(1) M(-)(1) A(-)(2) [Myers, J., Pace, N., and Scholtz, M. (1995) Protein Sci. 4, 2138]. These results indicate that urea titration can be used to measure both the free energy and the magnitude of an RNA folding transition.

Base Sequence↗
PubMed1983

Thiolated nucleotides in yeast transfer RNA.

By culturing Saccharomyces cerevisiae in growth medium containing Mg35SO4, we have determined the extent and variation of tRNA thiolation in this yeast. We find that 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U)1 is the major, if not only, thiolated derivative in S. cerevisiae tRNA. In addition, a comparison of the chromatographic mobility of mcm5s2Up on cellulose thin layers with those reported for unknown uridine derivatives found in purified yeast tRNA digests, leads to the conclusion that at least two of these tRNAs contain this modification.

RNA, Transfer↗
PubMed1986

Transfer ribonucleic acid populations in concanavalin-A-stimulated bovine lymphocytes.

Transfer RNA isolated from lymphocytes stimulated by concanavalin A and that from resting cells were compared with respect to amino-acid acceptance, integrity of the CCA-terminus, extent of modification and isoacceptor distribution. Following growth stimulation the overall amino-acid acceptance of the tRNA is elevated, in particular the relative acceptor activities for threonine and arginine are increased. The reduced acceptor activity of the tRNA from the quiescent cells is not due to a preferential degradation of the CCA-end, since it persists even in the presence of ATP(CTP):tRNA nucleotidyltransferase. We therefore conclude that this reduced activity is caused by structural differences of the tRNAs. The content of modified nucleotides in newly synthesized tRNA from lymphocytes cultured in the presence and absence of concanavalin A was determined. tRNA from resting cells was found to be undermodified with respect to pseudouridine and dihydrouridine. Upon monitoring the tRNA isoacceptor distribution by affinity chromatography on immobilized elongation factor Tu and subsequent two-dimensional gel electrophoresis, a preferential synthesis of particular lysine- and threonine-accepting tRNAs was observed upon mitogenic stimulation. Evidently, a specific tRNA population is needed by the proliferating cells. These results are discussed in view of the hypothesis that the commitment of lymphocytes to proliferation is at least in part under translational control.

Animals↗
PubMed1982

Conserved 5S rRNA complement to tRNA is not required for protein synthesis.

The notion that tRNA and 5S rRNA interact through evolutionarily conserved complementary sequences has been tested by nucleolytic modification of the 5S rRNA, using the modified rRNA to reconstitute the large ribosomal subunit, and assaying for poly(uridylic acid)-directed polyphenylalanine synthesis. The 5S rRNA sequence C-G-A-A (residues 43-46) and several residues surrounding it are not essential for protein synthesis.

Base Sequence↗
PubMed1999

Selective detection of ribose-methylated nucleotides in RNA by a mass spectrometry-based method.

Post-transcriptional methylation of ribose at position O-2' is one of the most common and conserved types of RNA modification. Details of the functional roles of these methylations are far from clear, although in tRNA they are involved at position 34 in regulation of codon recognition and in eukaryotic rRNAs they are required for subunit assembly. Experimental difficulties in the mapping of ribose methylations increase with RNA molecular size and the complexity of mixtures resulting from nuclease digestion. A new and relatively rapid approach based on tandem mass spectrometry is described in which any of four ion reaction pathways occurring in the mass spectrometer can be monitored which are highly specific for the presence of 2'-O -methylribose residues. These pathways emanate from further dissociation of ribose-methylated mononucleotide (Nmp) ions formed in the electrospray ionization region of the mass spectrometer to then form the base, methylribose phosphate or PO(3)(-)anions. The mass spectrometer can be set for detection of generic ribose methylation (Nm) in oligonucleotides, selectively for each of the common methylated nucleo-sides Cm, Gm, Am or Um or for specific cases in which the base or sugar is further modified. By direct combination of mass spectrometry with liquid chromatography the method can be applied to analysis of complex mixtures of oligonucleotides, as for instance from synthetic or in vitro reaction mixtures or from nuclease digests of RNA. An example is given in which the single ribose-methylated nucleoside in Escherichia coli 16S rRNA (1542 nt), N(4),O-2'-dimethylcytidine, is detected in 25 pmol of a RNase T1 digest and localized to the fragment 1402-CCCGp-1405 in a single 45 min analysis.

Escherichia coli↗
PubMed1978

Reaction of tRNAPhe from yeast with 1-fluoro-2,4-dinitrobenzene. Attachment sites of the potential antigenic-determining 2,4-dinitrophenyl residues.

The reaction of 1-fluoro-2,4-dinitrobenzene with tRNAPhe from yeast, for the introduction of antigenic-determining 2,4-dinitrophenyl residues into tRNA, took place only at adenosine residues in tRNAPhe. After reaction at pH 8.0 and 50 degrees C two kinds of products were detected: one was ribose-modified adenosine which was derived from the 3' terminus of tRNA, and the other was base-modified adenosine. The sites and extent of the modification of each particular adenosine residue of tRNAPhe were determined as follows: 5 (6% modified), 31 (2%), 35 (36%), 67 (5%), and 76 (51%). Thus mainly the terminal adenosine and one adenosine in the anticodon loop bear the 2,4-dinitrophenyl residue.

Base Sequence↗
PubMed1987

Ribosomal RNA methylation in Mycobacterium smegmatis SN2.

Ribosomal RNA (rRNA) from a fast growing nonpathogenic strain of mycobacteria, Mycobacterium smegmatis SN2, was analyzed for the presence of minor nucleotides. Of the sixteen modified nucleotides detected, the identity of twelve has been established and their molar ratios were determined. These nucleotides include m1A, m2A, m6A, m6(2)A, m7G, m5C, rT, CmpC, CmpG, GmpG, UmpG and UmpU. The distinct features of the mycobacterial rRNA modifications include: (i) relatively substantial level of methylation, a feature distinct from that of the tRNA species which are unique in being under methylated in these bacteria, (ii) N1 methyl adenine representing the bulk of the modified bases, (iii) the lack of ribose methylation on any two successive nucleotides, and (iv) the presence of N6,N6-dimethyl adenosines, which are the target sites of the antibiotic kasugamycin, although the bacterial growth is insensitive to the drug.

Carbon Radioisotopes↗
PubMed1980

The nucleotide sequence of two bovine lens phenylalanine tRNAs. Possible activation of a new phenylalanine tRNA gene during differentiation of lens cells.

Bovine lens fiber cells contain two major isoacceptors of phenylalanine tRNA, tRNA1Phe and tRNA2Phe whereas lens epithelial cells contain mainly tRNA2Phe (Ortwerth, B.J,, Yonuschot, G.R., Heidlege, J.F., Chu-Der, O.M.Y., Juarez, D., and Hedgcoth, C. (1975) Exp. Eye Res. 20, 417-426). Thus, the induction of a new species of tRNAPhe accompanies the differentiation of lens cells in vivo. These two species of tRNAPhe have been purified and their sequences have been analyzed. The results of the analyses show that the sequence of tRNA2Phe is identical with beef liver tRNAPhe while tRNA1Phe differs from tRNA2Phe by a guanosine to adenosine substitution at position 57. Therefore, the T psi CG sequence in the ribosylthymine loop of tRNA2Phe is replaced by T psi CA in tRNA1Phe, This observation demonstrates a case in which a change in sequence of tRNA isoacceptors accompanies cell development rather than changes in the level of post-transcriptional modification as noted in other systems (reviewed by Osterman L.A. (1979) Biochimie (Paris) 61, 323-342). The conversion of guanosine to adenosine in the ribosylthymine loop of lens tRNA1Phe suggests a possible activation of a new tRNAPhe gene during the differentiation of lens cells, although the possibility that this replacement may be catalyzed by a transglycosylase type enzyme has not been ruled out.

Animals↗
PubMed2006

Anticodons, frameshifts, and hidden periodicities in tRNA sequences.

Fourier analysis of the short-range periodicities for the complete set of sequences coding for tRNA genes in genome of Bacillus subtilis proves that periodicities with periods p = 2, 3, 4, and 6 sites are the inherent properties of tRNAs. The related periodicities should be understood in a broad statistical sense and their identifying needs the elaborate statistical methods. To improve the statistics, the analysis of significant periodicities was performed for the binary R-Y, S-W, and K-M sequences. Generally, such short-range periodicities are produced via biased positioning of particular nucleotides rather than via the tandem multiplication and subsequent modifications of repeats, though the latter mechanism may also be realized. Quasi-coherently piercing long segments of tRNA, the short-range periodicities create the effective long-range structural coupling between the acceptor stem and the anticodon loop and may participate in the mechanisms of molecular recognition. The periodicities with p = 2 and 4 provide the natural ground for the translation with spontaneous or programmed frameshifting and are present in tRNAs decoding the most frameshift-prone codons. The observation of short-range periodicities suggests that the mechanisms of amino-acylation of tRNAs and codon-anticodon pairing are not independent. Their study may also provide the important information related to the origin and evolution of the genetic code.

Anticodon↗
PubMed2002

rRNA modifications and ribosome function.

The development of three-dimensional maps of the modified nucleotides in the ribosomes of Escherichia coli and yeast has revealed that most (approximately 95% in E. coli and 60% in yeast) occur in functionally important regions. These include the peptidyl transferase centre, the A, P and E sites of tRNA- and mRNA binding, the polypeptide exit tunnel, and sites of subunit-subunit interaction. The correlations suggest that many ribosome functions benefit from nucleotide modification.

Binding Sites↗
PubMed2003

Roles of 5-substituents of tRNA wobble uridines in the recognition of purine-ending codons.

Many tRNA molecules that recognize the purine-ending codons but not the pyrimidine-ending codons have a modified uridine at the wobble position, in which a methylene carbon is attached directly to position 5 of the uracil ring. Although several models have been proposed concerning the mechanism by which the 5-substituents regulate codon-reading properties of the tRNAs, none could explain recent results of the experiments utilizing well-characterized modification-deficient strains of Escherichia coli. Here, we first summarize previous studies on the codon-reading properties of tRNA molecules with a U derivative at the wobble position. Then, we propose a hypothetical mechanism of the reading of the G-ending codons by such tRNA molecules that could explain the experimental results. The hypothesis supposes unconventional base pairs between a protonated form of the modified uridines and the G at the third position of the codon stabilized by two direct hydrogen bonds between the bases. The hypothesis also addresses differences between the prokaryotic and eukaryotic decoding systems.

Anticodon↗
PubMed1999

Methanopyrus kandleri glutamyl-tRNA reductase.

The initial reaction of tetrapyrrole formation in archaea is catalyzed by a NADPH-dependent glutamyl-tRNA reductase (GluTR). The hemA gene encoding GluTR was cloned from the extremely thermophilic archaeon Methanopyrus kandleri and overexpressed in Escherichia coli. Purified recombinant GluTR is a tetrameric enzyme with a native M(r) = 190,000 +/- 10,000. Using a newly established enzyme assay, a specific activity of 0.75 nmol h(-1) mg(-1) at 56 degrees C with E. coli glutamyl-tRNA as substrate was measured. A temperature optimum of 90 degrees C and a pH optimum of 8.1 were determined. Neither heme cofactor, nor flavin, nor metal ions were required for GluTR catalysis. Heavy metal compounds, Zn(2+), and heme inhibited the enzyme. GluTR inhibition by the newly synthesized inhibitor glutamycin, whose structure is similar to the 3' end of the glutamyl-tRNA substrate, revealed the importance of an intact chemical bond between glutamate and tRNA(Glu) for substrate recognition. The absolute requirement for NADPH in the reaction of GluTR was demonstrated using four NADPH analogues. Chemical modification and site-directed mutagenesis studies indicated that a single cysteinyl residue and a single histidinyl residue were important for catalysis. It was concluded that during GluTR catalysis the highly reactive sulfhydryl group of Cys-48 acts as a nucleophile attacking the alpha-carbonyl group of tRNA-bound glutamate with the formation of an enzyme-localized thioester intermediate and the concomitant release of tRNA(Glu). In the presence of NADPH, direct hydride transfer to enzyme-bound glutamate, possibly facilitated by His-84, leads to glutamate-1-semialdehyde formation. In the absence of NADPH, a newly discovered esterase activity of GluTR hydrolyzes the highly reactive thioester of tRNA(Glu) to release glutamate.

Amino Acid Substitution↗
PubMed1995

Phosphorylation of elongation factor Tu prevents ternary complex formation.

The elongation factor Tu (EF-Tu) is a member of the GTP/GDP-binding proteins and interacts with various partners during the elongation cycle of protein biosynthesis thereby mediating the correct binding of amino-acylated transfer RNA (aa-tRNA) to the acceptor site (A-site) of the ribosome. After GTP hydrolysis EF-Tu is released in its GDP-bound state. In vivo, EF-Tu is post-translationally modified by phosphorylation. Here we report that the phosphorylation of EF-Tu by a ribosome associated kinase activity is drastically enhanced by EF-Ts. The antibiotic kirromycin, known to block EF-Tu function, inhibits the modification. This effect is specific, since kirromycin-resistant mutants do become phosphorylated in the presence of the antibiotic. On the other hand, phosphorylated wild-type EF-Tu does not bind kirromycin. Most interestingly, the phosphorylation of EF-Tu abolishes its ability to bind aa-tRNA. In the GTP conformation the site of modification is located at the interface between domains 1 and 3 and is involved in a strong interdomain hydrogen bond. Introduction of a charged phosphate group at this position will change the interaction between the domains, leading to an opening of the molecule reminiscent of the GDP conformation. A model for the function of EF-Tu phosphorylation in protein biosynthesis is presented.

Anti-Bacterial Agents↗