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U L RajBhandary

Publications and source records attributed to U L RajBhandary.

At least 19 recordsLinked to original sources
PubMed1992

Striking effects of coupling mutations in the acceptor stem on recognition of tRNAs by Escherichia coli Met-tRNA synthetase and Met-tRNA transformylase.

We measured kinetic parameters in vitro and directly analyzed aminoacylation and formylation levels in vivo to study recognition of Escherichia coli initiator tRNA mutants by E. coli Met-tRNA synthetase and Met-tRNA transformylase. We show that, in addition to the anticodon sequence, mutations in the "discriminator" base A73 also affect aminoacylation. An A73----U change has a small effect, but a change to G73 or C73 significantly lowers Vmax/Kappm for in vitro aminoacylation and leads to appreciable accumulation of uncharged tRNA in vivo. Significantly, coupling of the G73 mutation with G72, a neighboring-base mutation, results in a tRNA essentially uncharged in vivo. Coupling of C73 and U73 mutations with G72 does not have such an effect. Elements crucial for Met-tRNA transformylase recognition of tRNAs are located at the end of the acceptor stem. These elements include a weak base pair or a mismatch between nucleotides (nt) 1 and 72 and base pairs 2.71 and 3.70. The natures of nt 1 and 72 are less important than the fact that they do not form a strong Watson-Crick base pair. Interestingly, the negative effect of a C.G base pair between nt 1 and 72 is suppressed by mutation of the neighboring nucleotide A73 to either C73 or U73. The presence of C73 or U73 could destabilize the C1.G72 base pair at the end of an RNA helix. Thus, in some tRNAs, the discriminator base could affect stability of the base pair between nt 1 and 72 and thereby the structure of tRNA at the end of the acceptor stem.

Acyltransferases↗
PubMed1992

Role of methionine and formylation of initiator tRNA in initiation of protein synthesis in Escherichia coli.

We showed recently that a mutant of Escherichia coli initiator tRNA with a CAU-->CUA anticodon sequence change can initiate protein synthesis from UAG by using formylglutamine instead of formylmethionine. We further showed that coupling of the anticodon sequence change to mutations in the acceptor stem that reduced Vmax/Km(app) in formylation of the tRNAs in vitro significantly reduced their activity in initiation in vivo. In this work, we have screened an E. coli genomic DNA library in a multicopy vector carrying one of the mutant tRNA genes and have found that the gene for E. coli methionyl-tRNA synthetase (MetRS) rescues, partially, the initiation defect of the mutant tRNA. For other mutant tRNAs, we have examined the effect of overproduction of MetRS on their activities in initiation and their aminoacylation and formylation in vivo. Some but not all of the tRNA mutants can be rescued. Those that cannot be rescued are extremely poor substrates for MetRS or the formylating enzyme. Overproduction of MetRS also significantly increases the initiation activity of a tRNA mutant which can otherwise be aminoacylated with glutamine and fully formylated in vivo. We interpret these results as follows. (i) Mutant initiator tRNAs that are poor substrates for MetRS are aminoacylated in part with methionine when MetRS is overproduced. (ii) Mutant tRNAs aminoacylated with methionine are better substrates for the formylating enzyme in vivo than mutant tRNAs aminoacylated with glutamine. (iii) Mutant tRNAs carrying formylmethionine are significantly more active in initiation than those carrying formylglutamine. Consequently, a subset of mutant tRNAs which are defective in formylation and therefore inactive in initiation when they are aminoacylated with glutamine become partially active when MetRS is overproduced.

Amino Acids↗
PubMed1992

Escherichia coli B lacks one of the two initiator tRNA species present in E. coli K-12.

We show that the metY locus which specifies tRNA(2fMet) in Escherichia coli K-12 specifies tRNA(1fMet) in E. coli B. This conclusion is based on results of Southern blot analysis of E. coli B and K-12 DNAs and on polymerase chain reaction amplification, cloning, and sequencing of an approximately 200-bp region of DNA corresponding to the metY loci of E. coli B and E. coli K-12. We also show that the metY locus of E. coli B is transcriptionally active. E. coli strains transformed with the multicopy plasmid vector pUC19 carrying the metY locus of E. coli B overproduce tRNA(1fMet) in E. coli B and E. coli K-12 in contrast to strains transformed with pUC19 carrying the corresponding locus from E. coli K-12, which overproduce tRNA(2fMet).

Base Sequence↗
PubMed1991

Direct analysis of aminoacylation levels of tRNAs in vivo. Application to studying recognition of Escherichia coli initiator tRNA mutants by glutaminyl-tRNA synthetase.

We describe the use of a gel electrophoretic method for measuring the levels of aminoacylation in vivo of mutant Escherichia coli initiator tRNAs, which are substrates for E. coli glutaminyl-tRNA synthetase (GlnRS) due to an anticodon sequence change. Using this method, we have compared the effects of introducing further mutations in the acceptor stem, at base pairs 1:72, 2:71, and 3:70 and discriminator base 73, on the recognition of these tRNAs by E. coli GlnRS in vitro and in vivo. The effects of the acceptor stem mutations on the kinetic parameters for aminoacylation of the mutant tRNAs in vitro are consistent with interactions seen between this region of tRNA and GlnRS in the crystal structure of tRNA(Gln). GlnRS complex. Except for one mutant, the observed levels of aminoacylation of the mutant tRNAs in vivo agree with those expected on the basis of the kinetic parameters obtained in vitro. We have also measured the relative amounts of aminoacyl-tRNAs for the various mutants and their activities in suppression of an amber codon in vivo. We find that there is, in general, a good correlation between the relative amounts of aminoacyl-tRNAs and their activities in suppression.

Acylation↗
PubMed1991

Mutants of Escherichia coli initiator tRNA that suppress amber codons in Saccharomyces cerevisiae and are aminoacylated with tyrosine by yeast extracts.

We recently described mutants of Escherichia coli initiator tRNA that suppress amber termination codons (UAG) in E. coli. These mutants have changes in the anticodon sequence (CAU----CUA) that allow them to read the amber codon and changes in the acceptor stem that allow them to bind to the ribosomal aminoacyl (A) site. We show here that a subset of these mutants suppress amber codons in Saccharomyces cerevisiae and that they are aminoacylated with tyrosine by yeast extracts. Analysis of a number of mutants as substrates for yeast tyrosyl-tRNA synthetase has led to identification of the C1.G72 base pair and the discriminator base A73, conserved in all eukaryotic cytoplasmic and archaebacterial tyrosine tRNAs, as being important for recognition. Our results suggest that the C1.G72 base pair and the discriminator base, in addition to the anticodon nucleotides previously identified [Bare, L.A. & Uhlenbeck, O.C. (1986) Biochemistry 25, 5825-5830] as important in yeast tyrosyl-tRNA synthetase recognition, may comprise the critical identity determinants in yeast tyrosine tRNA.

Amino Acyl-tRNA Synthetases↗
PubMed1991

Structural and sequence elements important for recognition of Escherichia coli formylmethionine tRNA by methionyl-tRNA transformylase are clustered in the acceptor stem.

We show that the structure and/or sequence of the first three base pairs at the end of the amino acid acceptor stem of Escherichia coli initiator tRNA and the discriminator base 73 are important for its formylation by E. coli methionyl-tRNA transformylase. This conclusion is based on mutagenesis of the E. coli initiator tRNA gene followed by measurement of kinetic parameters for formylation of the mutant tRNAs in vitro and function in protein synthesis in vivo. The first base pair found at the end of the amino acid acceptor stem in all other tRNAs is replaced by a C.A. "mismatch" in E. coli initiator tRNA. Mutation of this C.A. to U:A, a weak base pair, or U.G., a mismatch, has little effect on formylation, whereas mutation to C:G, a strong base pair, has a dramatic effect lowering Vmax/Kappm by 495-fold. Mutation of the second basepair G2:C71 to U2:A71 lowers Vmax/Kappm by 236-fold. Replacement of the third base-pair C3:G70 by U3:A70, A3:U70, or G3:C70 lowers Vmax/Kappm by about 67-, 27-, and 30-fold, respectively. Changes in the rest of the acceptor stem, dihydrouridine stem, anticodon stem, anticodon sequence, and T psi C stem have little or no effect on formylation.

Acyltransferases↗
PubMed1991

Mutants of initiator tRNA that function both as initiators and elongators.

We describe the effect of mutations in the acceptor stem of Escherichia coli initiator tRNA on its function in vivo. The acceptor stem mutations were coupled to mutations in the anticodon sequence from CAU----CUA to allow functional studies on the mutant tRNAs in initiation and in elongation in vivo. We show that, with one exception, there is a good correlation between the kinetic parameters for formylation of the mutant tRNAs in vitro (preceding paper, Lee, C.P., Seong, B. L., and RajBhandary, U.L. (1991) J. Biol. Chem. 266, 18012-18017) and their activity in initiation in vivo. These results suggest an important role for formylation of initiator tRNA in its function in initiation, at least when it is aminoacylated with glutamine as is the case with the mutant tRNAs used here. Mutant tRNAs that have a base pair between nucleotides 1 and 72 at the top of the acceptor stem function as elongators, as analyzed by their ability to suppress an amber mutation in the E. coli beta-galactosidase gene. One of these mutants is also quite active in initiation. Thus, activities of a tRNA in initiation and elongation steps of protein synthesis are not mutually exclusive. Using a mRNA with two in frame UAG codons, we show that this mutant tRNA can both initiate protein synthesis from the upstream UAG and suppress the down-stream UAG. We discuss the potential use of tRNAs with such "dual" functions in tightly regulated expression of genes for proteins in E. coli.

Ampicillin↗
PubMed1991

Expression of the bacterioopsin gene in Halobacterium halobium using a multicopy plasmid.

Bacteriorhodopsin (bR) was expressed in Halobacterium halobium by using a multicopy plasmid containing the bop gene. The plasmid contains pGRB1, a 1.8-kilobase-pair plasmid; a 70-base-pair fragment from ISH11, a recently characterized insertion sequence; and a 1.6-kilobase-pair fragment carrying the bop gene from H. halobium S9. When transformed with this plasmid, a bop- insertion mutant of H. halobium yielded purple (Pum+) colonies. The insertion at the chromosomal bop locus remained intact in transformed cells, indicating that the plasmid bop gene was responsible for the Pum+ phenotype. bR was induced in early stationary phase in both wild-type and transformed cells. The final level of bR in transformed cells was 25-40% of that in wild type. The lower level of expression was presumably due to plasmid instability. Purple membrane purified from transformed strains had absorption and visible CD properties similar to wild type and contained bR in a hexagonal lattice with the same unit-cell dimension as wild type. The structure of bR from wild-type and transformed strains was identical at a resolution of 7.2 A. When reconstituted into vesicles, the purple membrane from wild-type and transformed strains showed similar light-dependent proton-pumping activity.

Bacteriorhodopsins↗
PubMed1979

Mapping and cloning of Neurospora crassa mitochondrial transfer RNA genes.

We have obtained collections of recombinant Escherichia coli plasmids containing restriction fragments of Neurospora crassa mitochondrial DNA cloned into pBR322. By hybridization of 32P end-labeled total mitochondrial tRNAs and seven different purified tRNAs to restriction digests of mitochondrial DNA and of recombinant plasmids carrying specific restriction fragments, we have located the tRNA genes on the mitochondrial DNA. We have found that the mitochondrial tRNA genes are present in two major clusters, one between the two ribosomal RNA genes and the second closely following the large rRNA gene. Only one of the two DNA strands within these clusters codes for tRNAs. All of the genes for the seven specific purified tRNAs examined--those for alanine, formylmethionine, leucine 1, leucine 2, threonine, tyrosine, and valine--lie within these clusters. Interestingly, the formylmethionine tRNA hybridizes to two loci within one of these gene clusters. We have obtained a fairly detailed restriction map of part of this cluster and have shown that the two "putative" genes for formylmethionine tRNA are not arranged in tandem but are separated by more than 900 base pairs and by at least two other tRNA genes, those for alanine and for leucine 1 tRNAs.

Base Composition↗
PubMed1979

Nucleotide sequence of the 5' terminus of satellite tobacco necrosis virus ribonucleic acid.

Treatment of the RNA of satellite tobacco necrosis virus (STNV) with phosphomonoesterase followed by heat denaturation and treatment with polynucleotide kinase in the presence of [gamma-32P]ATP yields a STNV [5'-32P]RNA containing a homogeneous 5' terminus. Analyses of this STNV [5'-32P]RNA yield the sequence of the first 42 nucleotides from the 5'terminus of STNV RNA. This nucleotide sequence contains the translation initiation AUG codon starting at position 30 from the 5' terminus as indicated by match of subsequent nucleotides with the genetic code assignments for the N-terminal amino acids of STNV coat protein in the 5'-terminal sequence ppAGUAAAGACAGGAAACUU-UACUGACUAACAUGGCAAAACAAC. An interesting feature of this sequence is its potential to form a hairpin loop structure involving perfect Watson-Crick base pairing between the first seven nucleotides and nucleotides at positions 16--22.

Base Sequence↗
PubMed1979

Nucleotide sequence at the 5' terminus of adenovirus 2 late messenger RNA.

One major late promoter is transcriptionally active for the majority of adenovirus 2 late RNA synthesis. Nucleotide sequences proximal to this promoter are spliced onto the 5' termini of all messenger RNAs generated from it. We report here the complete nucleotide sequence of the capped 5-'terminal undecanucleotide common to these late mRNAs. The sequence m7G5'ppp5'm6AmCUCUCUUCCGp shows striking similarities to the 5'-terminal sequences of the eukaryotic alpha- and beta-globin mRNAs and contains the sequence 5'CUUCCG3' complementary to a six-base sequence near the 3' terminus of most eukaryotic 18 S ribosomal RNAs. On the basis of this sequence and the finding that this same sequence is present at the 5' end of adenovirus 2 late nuclear RNA precursor, it should now be possible to locate the major late promotor within the adenoviral genomic DNA sequence.

Adenoviruses, Human↗
PubMed1979

Organization of tRNA and rRNA genes in N. crassa mitochondria: intervening sequence in the large rRNA gene and strand distribution of the RNA genes.

Through analysis of cloned fragments of N. crassa mitochondrial DNA, we have derived a physical map for the region of the mitochondrial genome which encodes the ribosomal RNAs and most of the tRNAs. We have located RNA genes on this map by hybridization of purified 32P end-labeled RNA probes, and our findings are as follows. First, the gene for the large ribosomal RNA contains an intervening sequence of approximately 2000 bp. Second, the genes for the small and large ribosomal RNAs are not adjacent, as previously reported, and the region between them contains a number of tRNA genes, including that for the mitochondrial tRNATyr, which is located close to the small rRNA gene on the same strand of the mitochondrial DNA. Third, there is a second cluster of tRNA genes on the mitochondrial DNA following the large ribosomal RNA gene, but there is no evidence for the presence of tRNA genes in the intervening sequence of the large ribosomal RNA. Fourth, hybridization of labeled ribosomal and transfer RNAs to the separated strands of a cloned 16 kbp DNA fragment covering this region indicates that the two ribosomal RNAs and most, if not all, of the mitochondrial tRNAs are encoded on one strand of the mitochondrial DNA.

Base Sequence↗
PubMed1979

Interesting and unusual features in the sequence of Neurospora crassa mitochondrial tyrosine transfer RNA.

The mitochondrial tyrosine tRNA from Neurospora crassa has been sequenced and found to have several interesting features: (i) It resembles prokaryotic rather than eukaryotic tyrosine tRNAs in that it possesses a large variable loop (loop III); moreover, it can be quantitatively aminoacylated by Escherichia coli tyrosyl-tRNA synthetase but not by yeast tyrosyl-tRNA synthetase. (ii) This tRNA differs from all tRNA's sequenced to date in lacking the A residue at position 14 and the constant purine residue at position 15, two nucleosides that have been found so far in loop I of all tRNA's and that have been implicated in base-base tertiary interactions, respectively, with the universal U residue at position 8 and the constant pyrimidine residue at the end of loop III. (iii) Unlike the N. crassa mitochondrial initiator tRNA, this tRNA contains the usual TpsiC sequence in loop IV and the highly conserved GG sequence in loop I common to other tRNAs.

Base Sequence↗
PubMed1979

The nucleotide sequence of the initiator tRNA from Drosophila melanogaster.

The nucleotide sequence of Drosophila melanogaster methionine tRNAi was determined to be: pA-G-C-A-G-A-G-U-m1G-m2G-C-G-C-A-G-U-G-G-A-A-G-C-G-U-m2G-C-U-G-G-G-C-C-C-A-U-t6A-A-C-C-C-A-G-A-G-m7G-D-m5C-C-C-G-A-G-G-A-U-C-G-m1A-A-A-C-C-U-U-G-C-U-C-U-G-C-U-A-C-C-A(OH). It differs from vertebrate initiator tRNAs in only 6 out of 75 positions.

Animals↗