tRNA Trp Research Articles

Identification of RNA molecules which contain 5 S ribosomal RNA and transfer RNA in an RNAase E − RNAase P − double mutant strain of Escherichia coli

Mutant strains defective in the two processing enzymes RNAase E and RNAase P were constructed. In such strains an 11 S molecule was found from which it was possible to process in vitro precursor 5 S ribosomal RNA as well as tRNA 1 Asp and tRNA 3 Gly. From another somewhat more slowly migrating molecule precursor 5 S rRNA, tRNA 1 Asp, and tRNA Trp were processed. These molecules correspond to the distal parts of rRNA operons. We conclude that some tRNAs which are distal to rRNA genes are cotranscribed from the same promoters as rRNAs, and are processed by ribonuclease P.

Structural changes in the glutamine-chargeable Escherichia coli transfer RNA Trp produced by chemical modification with sodium bisulfite

Glutamine-mischargeable tRNA produced by sodium bisulfite-treated Escherichia coli tRNA Trp was isolated by dihydroxyboryl-cellulose affinity column chromatography. This tRNA was shown to have dual specificity for tryptophan and glutamine, and, when charged with either amino acid, bound to ribosomes in response to the non-sense codon UAG but not in response to the tryptophan codon UGG. The results were consistent with the reported properties of Su 7 + tRNA. The bisulfite-treated tRNA Trp migrated as two bands during polyacrylamide gel electrophoresis. The faster moving band (band I) coincided with that of untreated tRNA Trp. The slower moving band (band II) coincided with the glutamine-chargeable tRNA Trp. Su 7 + tRNA behaved like band II tRNA upon gel electrophoresis. Nucleotide sequence analysis showed that a cytidine—uridine transition occurred at the 1st or the 2n position of the anticodon of band II tRNA. Band I and band II tRNAs differed from each other in their thermal melting profiles. It is suggested that the single base change in the anticodon is responsible for the altered conformation of band II tRNA.

The effect of an Escherichia coli regulatory mutation on transfer RNA structure

The trpX mutation in Escherichia coli reduces trp operon attenuation in strains carrying wild-type tRNA Trp. The trpX − phenotype is alleviated (attenuation is restored) in UGA-suppressor tRNA Trp-carrying strains (Yanofsky & Soll, 1977). The tRNA from various trpX − strains was characterized biochemically. Sequence analyses of wild-type tRNA Trp and UGA suppressor tRNA Trp, both derived from trpX − strains, reveal an unmodified A in the position (adjacent to the anticodon) normally occupied by the hypermodified base ms 2i 6A. In addition, several tRNAs from trpX − cells were characterized by RPC-5 column chromatography. We find that only tRNAs normally having ms 2i 6A exhibit altered elution profiles when compared to the homologous tRNAs from trpX − cells. Introduction of the UGA suppressor into trpX − cells does not restore normal Chromatographic behavior. These results suggest that the trpX gene product is necessary for the synthesis of ms 2i 6A. Thus, we propose that miaA (for the first gene involved in ms 2 i 6 A synthesis) replaces the trpX designation. The results reported here are discussed with regard to a model proposed by Lee & Yanofsky (1977) in which efficient translation of the tandem trp codons in the leader sequence RNA is required for normal attenuation of the trp operon.

New procedure of high-voltage electrophoresis in polyacrylamide gel and its application to the sequencing of nucleic acids

Fractionation of nucleic acids and their fragments with polyacrylamide gel has been widely applied in sequencing of nucleic acids. Although the conditions of electrophoresis for this purpose have previously been suggested, we have found that polyacrylamide gel electrophoresis at 5000 V (100 V/cm) is possible and effective. An apparatus consisting of a horizontal thermostated plate is used to remove the heat which was formed during the electrophoretic process. The techniques for loading samples on the horizontal thin gel and the procedure for high-voltage gel electrophoresis are described and illustrated by the fractionation of the spleen phosphodiesterase partial digest of tRNA 2a Val as well as by the RNA synthesis by RNA polymerase from E. coli with poly[d(A-T)] as template in the presence of “terminator,” 3′- O-methyluridine 5′-triphosphate. This same technique was used for electrophoresis of oligonucleotides on acetylcellulose and was incorporated into a two-dimensional system which was demonstrated by fingerprinting of the guanylo-RNase digest of tRNA Trp from baker's yeast. In the third part of the article a simple technique for the electric trapping of nucleic acids or their fragments from a slab gel on a DEAE-paper sheet is presented.

Recognition of tRNATrp by Initiation Factors from Escherichia coli

Binding of acetyl or formyltryptophanyl-tRNA Trp from Escherichia coli or beef liver to E. coli ribosomes is strongly stimulated by E. coli initiation factors and requires GTP. The N-acylated tryptophan is puromycin reactive. Polypeptide chain initiation with acetyltryptophan dependent on poly(U,G) has been demonstrated and is highly dependent on added initiation factors. tRNA Trp appears, therefore, to share some structural features with tRNAfMet of significance to the process of polypeptide chain initiation.

In vitro transcription and processing of a yeast tRNA gene containing an intervening sequence

A gene for Saccharomyces cerevisiae tRNA Trp has been sequenced which contains an intervening sequence of 34 bp (H.S. Kang and J. Abelson, unpublished results). The mutant yeast strain ts-136 accumulates a precursor to tRNA Trp which contains mature ends and is colinear with the tRNA Trp gene. A nuclear extract from Xenopus oocytes is capable of supporting transcription of the tRNA Trp gene contained on plasmid pBR313. The products are precursor tRNAs which contain the intervening RNA sequence. The Xenopus extract accurately splices the precursor transcript to mature-sized tRNA Trp.

Primer tRNA Trp enhances the inhibition of avian myeloblastosis virus reverse transcriptase by pyridoxal-5′-phosphate

In the presence of tRNA Trp the inhibition of avian myeloblastosis virus reverse transcriptase by pyridoxal-5′-phosphate is greatly enhanced. This effect of tRNA is specific. While tRNA beef Trp (the primer of the in vitro DNA synthesis with 35 S viral RNA as template) gives the maximal effect, tRNA beef Val will not affect the enzyme activity. In the presence of tRNA beef Trp two additional lysines are titrated with pyridoxal-5′-phosphate as measured by reduction of the enzyme-pyridoxal-5′-phosphate complex with tritiated NaBH 4. The effect of tRNA is dependent on the presence of the β subunit of avian myeloblastosis virus reverse transcriptase, since the enzymatic activity of the α subunit, although inhibited by pyridoxal-5′-phosphate, is not affected by tRNA.

Effect of photochemical crosslink S 4U(8)-C(13) on suppressor activity of su + tRNA Trp from Escherichia coli

Readthrough in vitro of the Qβ coat protein terminator codon UGA has been used as an assay for suppression by UGA-suppressor tRNA Trp. When the tRNA is covalently crosslinked between 4-thiouracil(8) and cytosine(13) by irradiation at 334 nm, it is found that UGA suppression by this assay is reduced to the low level characteristic of the wild type tRNA Trp. In contrast, crosslinking has little effect on incorporation of tryptophan in response to UGG codons. Thus, incorporation of tryptophan during translation of R17 messenger RNA is unaffected by photochemical crosslinking. Furthermore, dilution experiments using R17 mRNA in which tryptophan incorporation is dependent on precharged suppressor Trp-tRNA show that the crosslinked species competes well with non-irradiated tRNA. These results further emphasize the influence on tRNA-ribosome interactions of the region in tRNA around the dihydrouridine arm, where the mutation, in the suppressor is found and the photochemical crosslink is introduced.

Deletion analysis of the expression of rRNA genes and associated tRNA genes carried by a lambda transducing bacteriophage

Transducing phage lambdailv5 carries genes for rRNA's, spacer tRNA's (tRNA1 Ile and tRNA1B Ala), and two other tRNA's (TRNA1 Asp and tRNA Trp). We have isolated a mutant of lambdailv5, lambdailv5su7, which carries an amber suppressor mutation in the tRNA Trp gene. A series of deletion mutants were isolated from the lambdailv5su7 phage. Genetic and biochemical analyses of these deletion mutants have confirmed our previous conclusion (E. A. Morgan, T. Ikemura, L. Lindahl, A. M. Fallon, and M. Nomura, Cell 13:335--344, 1978) that the genes for tRNA1 Asp and tRNA Trp located at the distal end of the rRNA operon (rrnC) are cotranscribed with other rRNA genes in that operon. In addition, these deletions were used to define roughly the physical location of the promoter(s) of the rRNA operon carried by the lambdailv5su7 transducing phage.

Blue dextran Sepharose chromatography of the tryptophanyl-tRNA synthetase of E.coli: a potential application for the purification of the enzyme

E. coli tryptophanyl-tRNA synthetase can form a complex with Blue-dextran Sepharose, in the presence or in the absence of Mg++. In its absence, the complex is dissociated by either ATP or cognate tRNATrp. However, in the presence of Mg++, only tRNATrp can dissociate the complex whereas ATP has no effect. E. coli total tRNA or tRNAMet, at the same concentration, cannot displace the synthetase from the complex. It is suggested that the Blue-dextran binds to the synthetase through its tRNA binding domain. This hypothesis is supported by previous findings with polynucleotide phosphorylase showing that Blue-dextran Sepharose can be used in affinity chromatography to recognize a polynucleotide binding site of the protein. The selective elution by its cognate tRNA of Trp-tRNA synthetase bound to Blue-dextran Sepharose provides a rapid and efficient purification of the enzyme. Examples of other synthetases and nucleotidyl transferases are also discussed.

18] Tryptophanyl-tRNA synthetase from beef pancreas

This chapter describes the isolation of tryptophanyl-tRNA synthetase [L-tryptophan: tRNA Trp ligase (AMP-forming), EC 6.1.1.2] from beef pancreas. The purification procedure comprises of five steps and yields about 130 mg of the enzyme per kilogram of tissue. The yield of the enzyme is considerably higher than that reported by others. Various properties and inhibitors of the given enzyme are described as well. From the mentioned data on beef pancreas tryptophanyl-tRNA synthetase it is concluded that this enzyme is characterized enzymologically rather intensively. Development of primary and three-dimensional structure investigations in the nearest future is expected because of the convenient and rapid procedure for enzyme purification. The availability of the specific inhibitors of different chemical nature makes it possible to evaluate various functional groups involved in the formation of active center. The mechanism of enzymic action needs to be investigated by means of fast kinetic methods.

Primary structure of bovine liver tRNA Trp

Purified tRNA Trp from bovine liver, accepting 1700 pmol tryptophan per A 260 nm unit, was completely digested with pancreatic ribonuclease and T 1 ribonuclease. The sequences of the resulting oligonucleotides were determined and the primary structure of the tRNA was deduced. These analyses showed numerous incomplete post-transcriptional modifications, and several positions heterogenously occupied by two different nucleotides, which lead us to think that in bovine liver there exist a mixture of several tRNA Trp.

Influence of Magnesium on the Steady‐State‐Derived Order of Substrate Addition and Product Release in tRNA Trp Aminoacylation by Beef Pancreas Tryptophan: tRNA Ligase

The order of the partial reactions in the aminoacylation of tRNATrpbeef by tryptophan : tRNA ligase from beef pancreas has been explored by measurements of intial velocity and product inhibition according to the analytical framework described by Cleland [Biochim. Biophys. Acta. 67, 104– 137, 173– 187, 188– 196 (1963)]. The kinetic patterns are suggestive of a ping‐pong mechanism (bi‐uni uni‐bi). The oreder of addition of tryptophan and ATP that one can derive is crucially dependent on the conditions of magnesium concentration used. Random fixation of tryptophan and ATP is observed in studies performed at catalytically optimal magnesium concentration. Linear‐ordered fixation of the two substrates, ATP binding first, is observed under conditions where magnesium concentration is in excess, which are not catalytically optimal. The analysis of the inhibition patterns taking into account the calculated concentrations of teh main active species Mg · ATP2− and Mg · P2O2−7 favors the random substrate‐binding mechanism derived under optimal magnesium conditions. Under both conditions, adjusted and fixed magnesium, the release of products, AMP and tryptophanyl‐tRNATrpbeef is, ordered, AMP released first.The change in the product nhibition patterns of aminoacylation of tRNATrpbeef by beef pancreas tryptophan: tRNA ligase under different magnesium concentration conditions casts some doubt on the significance of the comparison of mechanisms derived for amino‐acid: tRNA ligases of different sources.

Alkylation of tRNA Trp in a complex with tryptophanyl-tRNA synthetase

FEBS LettersVolume 90, Issue 1 p. 103-106 Full-length articleFree Access Alkylation of tRNATrp in a complex with tryptophanyl-tRNA synthetase V.V. Vlassov, V.V. Vlassov Institute of Organic Chemistry, Siberian Division of the USSR Academy of Sciences, 630090 Novosibirsk USSR Institute of Molecular Biology, USSR Academy of Sciences, 117312 Moscow, USSRSearch for more papers by this authorV.E. Tchizhikov, V.E. Tchizhikov Institute of Organic Chemistry, Siberian Division of the USSR Academy of Sciences, 630090 Novosibirsk USSR Institute of Molecular Biology, USSR Academy of Sciences, 117312 Moscow, USSRSearch for more papers by this author V.V. Vlassov, V.V. Vlassov Institute of Organic Chemistry, Siberian Division of the USSR Academy of Sciences, 630090 Novosibirsk USSR Institute of Molecular Biology, USSR Academy of Sciences, 117312 Moscow, USSRSearch for more papers by this authorV.E. Tchizhikov, V.E. Tchizhikov Institute of Organic Chemistry, Siberian Division of the USSR Academy of Sciences, 630090 Novosibirsk USSR Institute of Molecular Biology, USSR Academy of Sciences, 117312 Moscow, USSRSearch for more papers by this author First published: June 01, 1978 https://doi.org/10.1016/0014-5793(78)80307-XCitations: 6AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume90, Issue1June 01, 1978Pages 103-106 ReferencesRelatedInformation

Avian retrovirus RNA-directed DNA synthesis: Transcription at the 5′ terminus of the viral genome and the functional role for the viral terminal redundancy

The size and structure of DNA synthesized on 35 S RNA·tRNA trp template primer complexes in reconstructed reactions by the avian retrovirus RNA-directed DNA polymerase in vitro have been elucidated. Under standard enzymatic reaction conditions three major species of DNA are initiated on tRNA trp at the 5′ end of the viral genome. Two of these are single stranded in nature and exhibit lengths of 70 and 100 nucleotides. The third is hairpin in structure and is 185 nucleotides in length. A precursor-product relationship has been demonstrated between DNA 70, DNA 100, and the hairpin DNA 185 species. Furthermore, it appears that hairpin DNA 185 is formed during DNA synthesis rather than after DNA synthesis as a consequence of the intramolecular folding of complementary sequences present in the DNA 185 transcript. Finally, we present data indicating that, in addition to conversion of DNA 100 to hairpin DNA, the DNA 100 species can attach to the 3′ end of the viral genome presumably by base pairing with the terminally redundant sequences at the 3′ terminus to provide a primer for the continuation of DNA transcription at this end of the viral RNA. These data provide a functional role in proviral DNA synthesis for the terminally repeated genomic sequences.

In vitro transcription of reconstituted 35S RNA·tRNA trp template·primer complexes by the avian oncornavirus DNA polymerase Effect of temperature on the size of the DNA transcripts

The synthesis of long DNA transcripts from avian sarcoma virus 35S RNA·tRNA trp template·primer complexes in reconstituted reactions in vitro is temperature dependent. At 15° DNA transcripts accumulate that are 100 nucleotides in length (DNA 100), whereas at 30° DNA transcripts ranging to 3000 nucleotides in length (DNA >100) are synthesized from these template·primer complexes. By hybridization analysis we have demonstrated that DNA 100 synthesized at 15° contains nucleotide sequences complementary to the 5′ end of the viral RNA genome whereas longer DNA transcripts synthesized at 30° contain nucleotide sequences complementary to the 3′ terminus of the viral genome. Pulse-chase experiments indicate that the 5′-located DNA 100 transcripts are precursors of the larger DNA transcripts. These data are consistent with the elongation of 5′ terminally located DNA transcripts at the 3′ end of the viral genome.

Some rRNA operons in E. coli have tRNA genes at their distal ends

We have previously isolated seven rRNA operons on plasmids or λ transducing phages and identified various tRNAs encoded by these operons. Each of the seven operons has one of two different spacer tRNA gene arrangements between the genes for 16S and 23S rRNA: either tRNA 2 Glu or both tRNA 1 Ile and tRNA 1B Ala genes. In addition, various tRNA genes are located at or near the distal ends of rRNA operons. In particular, genes for tRNA Trp and tRNA 1 Asp are located at the distal end of rrnC at 83 min on the E. coli chromosome. Experiments with various hybrid plasmids, some of which lack the rRNA promoter, have now demonstrated that this promoter is necessary for expression of the distal tRNA genes. Rifampicin run-out experiments have also provided evidence that the tRNA Trp gene is located farther from its promoter than the spacer tRNA gene or the 5S RNA gene. These results confirm the localization of genes for tRNA Trp and tRNA 1 Asp at the distal end of rrnC and strongly suggest that they are co-transcribed with the genes for 16S, tRNA 2 Glu, 23S and 5S RNA. Other such distal tRNAs have been identified, and it is suggested that they too are part of rRNA operons.

Nucleotide sequence of Rous sarcoma virus RNA at the initiation site of DNA synthesis: The 102nd nucleotide is U

The T 1 oligonucleotide in the genome Rous sarcoma virus (RSV) that corresponds to the initiation site of DNA synthesis in vitro was identified by hybridization of genome RNA with RSV strong stop DNA (the initial 101-nucleotide long fragment synthesized in endogenous reactions) and partially sequenced. The sequence of (C 2, U 2) A-U-U-U-G found corresponds to the d(A-A-T-G-A-A-G) sequence at the 5′ end of the DNA product plus the CA-OH sequence at the 3′ end of the tRNA Trp primer. Therefore the nucleotide opposite the terminal A of the primer is the complementary U. Furthermore, no internal repetition of more than 30 nucleotides of the 5′ sequence could be detected.

Mutations affecting tRNA Trp and its charging and their effect on regulation of transcription termination at the attenuator of the tryptophan operon

A mutant altered in the sole structural gene for tRNA Trp produces sevenfold elevated levels of trp operon enzymes and messenger RNA. These increases are probably due to a reduced frequency of transcription termination at the attenuator region of the operon. A normal termination frequency is observed in this mutant, however, when conditions are imposed that should permit an increase in the extent of charging of the mutant tRNA Trp. Similarly, a mutant with an altered tryptophanyl-tRNA synthetase exhibits increased expression of the operon; this increase is also eliminated by conditions which should allow charging of a greater fraction of cellular tRNA Trp. These observations suggest that the extent of charging of tRNA Trp rather than the availability of tryptophan is the determinative factor in regulation at the attenuator. A second mutation which alters tRNA Trp, but which is not in the tRNA Trp structural gene, also increases expression of the operon. Conditions which should promote charging of this tRNA Trp do not overcome the regulatory defect. Analyses were performed with various trpT heterozygotes in an attempt to determine whether charged or uncharged tRNA Trp is the active species in the regulation of transcription termination at the attenuator. The findings obtained suggest that both species influence regulation at the attenuator.

Affinity labelling of tryptophanyl-transfer RNA synthetase

A double affinity-labelling approach has been developed in order to convert an oligomeric enzyme with multiple active centres into a single-site enzyme. Tryptophanyl-transfer RNA synthetase (EC 6.1.1.2) from beef pancreas is a symmetric dimer, α 2 An ATP analogue, γ-( p-azidoanilide)-ATP does not serve as a substrate for enzymatic aminoacylation of tRNA Trp but acts as an effective competitive inhibitor in the absence of photochemical reaction, with K 1 = 1 × 10 −3m ( K mfor ATP = 2 × 10 −4m). The covalent photoaddition of azido-ATP ‡ ‡ Abbriviations used: azido-ATP, γ-( p-azidoanilide)adenosine triphosphate; TRSase, tryptophanyl-tRNA synthetase (EC 6.1.1.2); R-Trp-tRNA, a residue of ambucilyl-tryptophanyl-tRNA covalently bound to the protein; Cl-R-Trp-tRNA, N-chlorambucilyl-tryptophanyl-tRNA; chlorambucil, γ- p-bis(2-chloroethyl)-aminophenyl-butyrie acid; -R-tryptophan, a residue of ambucilyl-tryptophan covalently bound to the protein; BDC, benzoylated diethylaminoethyl cellulose. results in complete loss of enzymatic activity in both the ATP-[ 32P]pyrophosphate exchange reaction and tRNA aminoacylation. ATP completely protects the enzyme against inactivation. However, covalent binding of azido-ATP is also observed outside the active centres. The difference between covalent binding of the azido-ATP in the absence and presence of ATP corresponds to 2 moles of the ATP analogue per mole of the enzyme. Two binding sites for tRNA Trp have been found from complex formation at pH 5.8 in the presence of Mg 2+. The two tRNA molecules bind, with K dis = 3.6 × 10 −8m and K dis = 0.9 × 10 −6m, respectively, pointing to a strong negative co-operativity between the binding sites for tRNA. N-chlorambucilyl-tryptophanyl-tRNA Trp and TRSase form a complex with K dis = 5.5 × 10 −8m at pH 5.8 in the presence of 10 m m-Mg 2+. This value is similar to the value of K dis for tryptophanyl-tRNA of 4.8 × 10 −8 m. Under the same conditions a 1:1 complex (in mol) is formed between the enzyme and Trp-tRNA or N-chlorambucilyl-Trp-tRNA. On incubation, a covalent bond is formed between N-chlorambucilyl-Trp-tRNA and TRSase; 1 mole of affinity reagent alkylates 1 mole of enzyme independently of the concentration of the modifier. The alkylation reaction is completely inhibited by the presence of tRNA Trp whereas the tRNA devoid of tRNA Trp does not affect the rate of alkylation. In the presence of either ATP or tryptophan, or a mixture of the two, the alkylation reaction is inhibited even though these ligands have no effect on the complex formation between TRSase and the tRNA analogue. Photoaddition of the azido-ATP completely prevents the reaction of the enzyme with the tRNA analogue, although the non-covalent complex formation is not affected. Exhaustive alkylation of TRSase partially inhibits the reaction of ATP [ 32P]pyrophosphate exchange and completely blocks the aminoacylation of tRNA Trp. Cleavage of the tRNA which is covalently bound to TRSase restores both the ATP-[ 32P]pyrophosphate exchange and aminoacylation activity. The TRSase which is covalently-bound to R-Trp-tRNA is able to incorporate only one ATP molecule per dimeric enzyme into the active centre. This doubly modified enzyme is completely enzymatically inactive. Removal of the tRNA residue from the doubly modified enzyme results in the formation of the derivative with one blocked ATP site. Therefore, a “single-site” TRSase may be generated either by alkylation of the enzyme with Cl-R-Trp-tRNA or after the removal of covalently bound tRNA from the doubly labelled protein. Tryptophanyl-tRNA synthetase containing blocked ATP and/or tRNA binding site(s) seems to bo a useful tool for investigation of negative co-operativity and may help in the elucidation of the structure function relationships between the active centres.