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U L Rajbhandary

Publications and source records attributed to U L Rajbhandary.

16 recordsLinked to original sources
PubMed1998

Initiator-elongator discrimination in vertebrate tRNAs for protein synthesis.

Initiator tRNAs are used exclusively for initiation of protein synthesis and not for the elongation step. We show, in vivo and in vitro, that the primary sequence feature that prevents the human initiator tRNA from acting in the elongation step is the nature of base pairs 50:64 and 51:63 in the TpsiC stem of the initiator tRNA. Various considerations suggest that this is due to sequence-dependent perturbation of the sugar phosphate backbone in the TpsiC stem of initiator tRNA, which most likely blocks binding of the elongation factor to the tRNA. Because the sequences of all vertebrate initiator tRNAs are identical, our findings with the human initiator tRNA are likely to be valid for all vertebrate systems. We have developed reporter systems that can be used to monitor, in mammalian cells, the activity in elongation of mutant human initiator tRNAs carrying anticodon sequence mutations from CAU to CCU (the C35 mutant) or to CUA (the U35A36 mutant). Combination of the anticodon sequence mutation with mutations in base pairs 50:64 and 51:63 yielded tRNAs that act as elongators in mammalian cells. Further mutation of the A1:U72 base pair, which is conserved in virtually all eukaryotic initiator tRNAs, to G1:C72 in the C35 mutant background yielded tRNAs that were even more active in elongation. In addition, in a rabbit reticulocyte in vitro protein-synthesizing system, a tRNA carrying the TpsiC stem and the A1:U72-to-G1:C72 mutations was almost as active in elongation as the elongator methionine tRNA. The combination of mutant initiator tRNA with the CCU anticodon and the reporter system developed here provides the first example of missense suppression in mammalian cells.

Acylation↗
PubMed1997

Intragenic suppression in tRNA: evidence for crosstalk between the D and the T stems.

We showed previously that introduction of two of the three unique features of Escherichia coli initiator tRNA onto an elongator methionine tRNA conferred significant activity in initiation. Surprisingly, introduction also of the third unique feature, the A11:U24 base pair in the D stem, resulted in total lack of accumulation of the mutant Mi:3 tRNA. We show here that the Mi:3 tRNA gene is transcribed efficiently in vitro. Processing of the Mi:3 precursor transcript shows, however, that both the precursor and the mature Mi:3 tRNA are unstable in E. coli extracts. To understand the basis of instability caused by the A11:U24 base pair in the elongator methionine tRNA background, we have isolated and characterized intragenic suppressor mutations in the tRNA that restore its function in translation initiation. Sequence changes in the T stem that convert the existing A51 x C63 mismatch to a base pair in the Mi:3 tRNA result in accumulation of the tRNAs in vivo. The initiation activity and in vivo levels of accumulation of these suppressors are in the order Mi:3/G51:C63 > Mi:3/A51:U63 >> Mi:3/G51.U63. These results show that the in vivo accumulation of a tRNA with A11:U24 base pair in the D stem depends upon a base pair between positions 51 and 63 in the T stem. Structural analysis in vitro of the Mi:3 and Mi:3/G51:C63 transcripts suggests that the Mi:3 tRNA is unable to adopt a stable tRNA-like conformation. Various considerations suggest that this is most likely due to a high entropic barrier to tertiary interactions, between the D and the T loops necessary for the formation of a stable tRNA structure.

Base Sequence↗
PubMed1996

Role of the three consecutive G:C base pairs conserved in the anticodon stem of initiator tRNAs in initiation of protein synthesis in Escherichia coli.

The three consecutive G:C base pairs, G29:C41, G30:C40, and G31:C39, are conserved in the anticodon stem of virtually all initiator tRNAs from eubacteria, eukaryotes, and archaebacteria. We show that these G:C base pairs are important for function of the tRNA in initiation of protein synthesis in vivo. We changed these base pairs individually and in combinations and analyzed the activities of the mutant Escherichia coli initiator tRNAs in initiation in vivo. For assessment of activity of the mutant tRNAs in vivo, mutations in the G:C base pairs were coupled to mutation in the anticodon sequence from CAU to CUA. Mutations in each of the G:C base pairs reduced activity of the mutant tRNA in initiation, with mutation in the second G:C base pair having the most severe effect. The greatly reduced activity of this C30:G40 mutant tRNA is not due to defects in aminoacylation or formulation of the tRNA or defects in base modification of the A37, next to the anticodon, which we had previously shown to be important for activity of the mutant tRNAs in initiation. The anticodon stem mutants are most likely affected specifically at the step of binding to the ribosomal P site. The pattern of cleavages in the anticodon loop of mutant tRNAs by S1 nuclease indicate that the G:C base pairs may be involved directly in interactions of the tRNA with components of the P site on the ribosome rather than indirectly by inducing a particular conformation of the anticodon loop critical for function of the tRNA in initiation.

Acylation↗
PubMed1990

Expression and function of a human initiator tRNA gene in the yeast Saccharomyces cerevisiae.

We showed previously that the human initiator tRNA gene, in the context of its own 5'- and 3'-flanking sequences, was not expressed in Saccharomyces cerevisiae. Here we show that switching its 5'-flanking sequence with that of a yeast arginine tRNA gene allows its functional expression in yeast cells. The human initiator tRNA coding sequence was either cloned downstream of the yeast arginine tRNA gene, with various lengths of intergenic spacer separating them, or linked directly to the 5'-flanking sequence of the yeast arginine tRNA coding sequence. The human initiator tRNA made in yeast cells can be aminoacylated with methionine, and it was clearly separated from the yeast initiator and elongator methionine tRNAs by RPC-5 column chromatography. It was also functional in yeast cells. Expression of the human initiator tRNA in transformants of a slow-growing mutant yeast strain, in which three of the four endogenous initiator tRNA genes had been inactivated by gene disruption, resulted in enhancement of the growth rate. The degree of growth rate enhancement correlated with the steady-state levels of human tRNA in the transformants. Besides providing a possible assay for in vivo function of mutant human initiator tRNAs, this work represents the only example of the functional expression of a vertebrate RNA polymerase III-transcribed gene in yeast cells.

Gene Expression↗
PubMed1989

Nuclear gene for mitochondrial leucyl-tRNA synthetase of Neurospora crassa: isolation, sequence, chromosomal mapping, and evidence that the leu-5 locus specifies structural information.

We have isolated and characterized the nuclear gene for the mitochondrial leucyl-tRNA synthetase (LeuRS) of Neurospora crassa and have established that a defect in this structural gene is responsible for the leu-5 phenotype. We have purified mitochondrial LeuRS protein, determined its N-terminal sequence, and used this sequence information to identify and isolate a full-length genomic DNA clone. The 3.7-kilobase-pair region representing the structural gene and flanking regions has been sequenced. The 5' ends of the mRNA were mapped by S1 nuclease protection, and the 3' ends were determined from the sequence of cDNA clones. The gene contains a single short intron, 60 base pairs long. The methionine-initiated open reading frame specifies a 52-amino-acid mitochondrial targeting sequence followed by a 942-amino-acid protein. Restriction fragment length polymorphism analyses mapped the mitochondrial LeuRS structural gene to linkage group V, exactly where the leu-5 mutation had been mapped before. We show that the leu-5 strain has a defect in the structural gene for mitochondrial LeuRS by restoring growth under restrictive conditions for this strain after transformation with a wild-type copy of the mitochondrial LeuRS gene. We have cloned the mutant allele present in the leu-5 strain and identified the defect as being due to a Thr-to-Pro change in mitochondrial LeuRS. Finally, we have used immunoblotting to show that despite the apparent lack of mitochondrial LeuRS activity in leu-5 extracts, the leu-5 strain contains levels of mitochondrial LeuRS protein to similar to those of the wild-type strain.

Amino Acid Sequence↗
PubMed1989

Regulation of the nuclear genes encoding the cytoplasmic and mitochondrial leucyl-tRNA synthetases of Neurospora crassa.

We show that the nuclear genes for the cytoplasmic and mitochondrial leucyl-tRNA synthetase (LeuRS) of Neurospora crassa are distinct in their encoded proteins, codon usage, mRNA levels, and regulation. The 4.2-kilobase-pair region representing the structural gene for cytoplasmic LeuRS and flanking regions has been sequenced. The positions of the 5' and 3' ends of mRNA and of a single 62-base-pair intron have been mapped. The methionine-initiated open reading frame encoded a protein of 1,123 amino acids and displayed a strong codon bias. Although cytoplasmic LeuRS shares with mitochondrial LeuRS some general features common to most aminoacyl-tRNA synthetases, there is little amino acid sequence similarity between them, mRNA levels for cytoplasmic LeuRS were much higher than those for mitochondrial LeuRS. This observation and the strong codon bias in the cytoplasmic LeuRS gene may contribute to a greater abundance of cytoplasmic LeuRS than mitochondrial LeuRS. The genes for cytoplasmic and mitochondrial LeuRS are regulated independently. The cytoplasmic LeuRS gene is regulated by the cross-pathway control system in N. crassa, which is analogous to general amino acid control in Saccharomyces cerevisiae. The cytoplasmic LeuRS mRNA levels are induced by amino acid starvation resulting from the addition of aminotriazole. Part of this increase is due to utilization of new transcription start sites. In contrast, the mitochondrial LeuRS gene is not induced by amino acid limitation. However, the mitochondrial LeuRS mRNA levels did increase dramatically upon inhibition of mitochondrial protein synthesis by chloramphenicol or ethidium bromide or in the temperature-sensitive strain leu-5 carrying a mutation in the mitochondrial LeuRS structural gene.

Amino Acid Sequence↗
PubMed1980

Recent developments in methods for RNA sequencing using in vitro 32P-labeling.

A variety of approaches that utilize in vitro 32P-labeling of RNA and of oligonucleotides in the sequence analysis of RNAs are described. These include 1) methods for 5'- and 3'- end labeling of RNAs; 2) end labeling and sequencing of oligonucleotides present in complete T1 RNase or pancreatic RNase digests of RNA; 3) use of random endonucleases, such as nuclease P1, for terminal sequence analysis of end labeled RNAs; and 4) use of base specific enzymes or chemical reagents in the sequence analysis of end-labeled RNAs. Also described is an approach to RNA sequencing, applied so far to tRNAs, which is based on partial and random alkaline cleavage of an RNA to generate a series of overlapping oligonucleotide fragments, all containing the original 3'-end of the RNA. Analysis of the 5'- end group of each of these oligonucleotides (following 5'-end labeling with 32P) provides the sequence of most of the tRNA. The above methods have been used to derive the sequences of several tRNAs, the ribosomal 5S and 5 x 8S RNAs, a viroid RNA, and large segments of both prokaryotic and eukaryotic ribosomal and messenger RNAs.

Autoradiography↗
PubMed1979

Nucleotide sequence of three isoaccepting lysine tRNAs from rabbit liver and SV40-transformed mouse fibroblasts.

The lysine isoacceptor tRNAs differ in two aspects from the majority of the other mammalian tRNA species: they do not contain ribosylthymine (T) in loop IV, and a 'new' lysine tRNA, which is practically absent in non-dividing tissue, appears at elevated levels in proliferating cells. We have therefore purified the three major isoaccepting lysine tRNAs from rabbit liver and the 'new' lysine tRNA isolated from SV40-transformed mouse fibroblasts, and determined their nucleotide sequences. Our basic findings are as follows. a) The three major lysine tRNAs (species 1, 2 and 3) from rabbit liver contain 2'-O-methylribosylthymine (Tm) in place of T. tRNA1Lys and tRNA2Lys differ only by a single base pair in the middle of the anticodon stem; the anticodon sequence C-U-U is followed by N-threonyl-adenosine (t6A). TRNA3Lys has the anticodon S-U-U and contains two highly modified thionucleosides, S (shown to be 2-thio-5-carboxymethyl-uridine methyl ester) and a further modified derivative of t6 A (2-methyl-thio-N6-threonyl-adenosine) on the 3' side of the anticodon. tRNA3Lys differs in 14 and 16 positions, respectively, from the other two isoacceptors. b) Protein synthesis in vitro, using synthetic polynucleotides of defined sequence, showed that tRNA2Lys with anticodon C-U-U recognized A-A-G only, whereas tRNA3Lys, which contains thio-nucleotides in and next to the anticodon, decodes both lysine codons A-A-G and A-A-A, but with a preference for A-A-A. In a globin-mRNA-translating cell-free system from ascites cells, both lysine tRNAs donated lysine into globin. The rate and extent of lysine incorporation, however, was higher with tRNA2Lys than with tRNA3Lys, in agreement with the fact that alpha-globin and beta-globin mRNAs contain more A-A-G than A-A-A- codons for lysine. c) A comparison of the nucleotide sequences of lysine tRNA species 1, 2 and 3 from rabbit liver, with that of the 'new' tRNA4Lys from transformed and rapidly dividing cells showed that this tRNA is not the product of a new gene or group of genes, but is an undermodified tRNA derived exclusively from tRNA2Lys. Of the two dihydrouridines present in tRNA2Lys, one is found as U in tRNA4Lys; the purine next to the anticodon is as yet unidentified but is known not be t6 A. In addition we have found U, T and psi besides Tm as the first nucleoside in loop IV.

Animals↗
PubMed1977

Studies on the sequence of the 3'-terminal region of turnip-yellow-mosaic-virus RNA.

A fragment representing the 3'-terminal 'tRNA-like' region of turnip yellow mosaic (TYM) virus RNA has been purified following incubation of intact TYM virus RNA with Escherichia coli 'RNase P'. This fragment, which is 112+3-nucleotides long has been completely digested with T1 RNase and pancreatic RNase and all the oligonucleotides present in such digests have been sequenced using 32P-end labelling techniques in vitro. The TYM virus RNA fragment is free of modified nucleosides and does not contain a G-U-U-C-R sequence. Using nuclease P1 from Penicillium citrinum, the sequence of 26 nucleotides from the 5' end and 16 nucleotides from the 3' end of this fragment has been deduced. The nucleotide sequence at the 5' end of the TYM virus RNA fragment indicates that this fragment includes the end of the TYM virus coat protein gene.

Base Sequence↗
PubMed1976

Nucleotide sequences at the 5'termini of rabbit alpha and beta globin mRNA.

The nucleotide sequences at the 5' termini of rabbit alpha and beta globin mRNAs have been determined. Periodate oxidation of globin mRNA followed by reduction with 3H-sodium borohydride and subsequent analysis of the 3H-labeled mRNA reveals the presence of the "cap" structure m7G5'ppp- blocking the 5' terminus. After periodate oxidation, beta elimination, and phosphomonoesterase treatment to remove the m7G5'ppp- "cap," the 5' end of globin mRNA was labeled with 32P using gamma-32P-ATP and T4 polynucleotide kinase. The 5'-32P-labeled alpha and beta globin mRNAs were then resolved from each other by polyacrylamide gel electrophoresis under denaturing conditions and sequenced separately. The 5' terminal nucleotide sequences determined are: alpha--m7G5'ppp5m6AmC(m)ACUUCUGG- BETA--M7G5'ppp5m6AmC(m) ACUUGCUUUUGACACAA Besides the m7G "cap" structure, the two sequences are identical for the first six nucleotides and then diverge. No initiator codon is present within the first ten nucleotides from the 5' end of the alpha globin mRNA, and the first nineteen nucleotides from the 5' end of beta globin mRNA.

Animals↗
PubMed1973

Absence of the sequence G-T-psi-C-G(A)- in several eukaryotic cytoplasmic initiator transfer RNAs.

The nucleotide sequence G-T-Psi-C-G(A)- has previously been found in every tRNA of known sequence that is active in protein biosynthesis. An exception to this generalization is the recently sequenced initiator tRNA from yeast cytoplasm. It is now reported that cytoplasmic initiator tRNAs from wheat germ, rabbit liver, and sheep mammary gland also lack the G-T-Psi-C-G(A)- sequence. Thus: (i) nucleoside composition analyses show the absence of T in all these tRNAs; (ii) analyses of oligonucleotide fragments produced by T1 ribonuclease show the absence not only of the T-Psi-C-G(A)- sequence, but also of U-Psi-C-G(A)- or U-U-C-G(A)- sequences in such digests. The absence of G-T-Psi-C-G(A)- in the eukaryotic cytoplasmic initator tRNAs is, therefore, not simply due to lack of enzymatic modification of U to T.

Adenosine↗
PubMed1973

Replacement of the sequence G-T-phi-C-G(A)- by G-A-U-C-G- in initiator transfer RNA of rabbit-liver cytoplasm.

Eukaryotic cytoplasmic initiator tRNAs lack the sequence G-T-Psi-C-G(A)-, which is in every tRNA of known sequence that is active in protein biosynthesis. In initiator tRNA of yeast cytoplasm, which is the only eukaryotic initiator tRNA of known sequence, this sequence is replaced by G-A-U-C-G-. We now report the sequence of a 30-nucleotide-long, 3'-terminal fragment of cytoplasmic initiator tRNA of rabbit liver obtained by specific cleavage of the tRNA at the site occupied by the modified nucleoside, 7-methyl guanosine. We show (i) that in rabbit-liver initiator tRNA also, the sequence G-T-Psi-C-G(A)- is replaced by G-A-U-C-G- and (ii) that the sequences of loop IV of both the yeast and rabbit-liver cytoplasmic initiator tRNAs are identical, A-U-C-G-m(1)-A-A-A-.

Adenine Nucleotides↗