The Nucleotide Sequences of Tyrosine Transfer RNAs of Escherichia coli

The nucleotide sequence G-T-Ψ-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-Ψ-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-Ψ-C-G(A)- sequence, but also of U-Ψ-C-G(A)- or U-U-C-G(A)- sequences in such digests. The absence of G-T-Ψ-C-G(A)- in the eukaryotic cytoplasmic initator tRNAs is, therefore, not simply due to lack of enzymatic modification of U to T.

“…Fingerprints of the radioactive fragments from E. coli tRNAIr ( Fig. 2A) are consistent with those obtained from tRNA,, labeled in vivo with 32p (29).…”

Section: Eukaryotic Cytoplasmic Initiator Transfer Rnas 1045supporting

confidence: 76%

Absence of the Sequence G-T-Ψ-C-G(A)- in Several Eukaryotic Cytoplasmic Initiator Transfer RNAs

Şi̇mşek¹,

Ziegenmeyer²,

Heckman³

et al. 1973

“…The infection of Escherichia coli by the phage 480 pSU3+ leads to the synthesis of tRNATYr (6). The final su3+ tRNATYr product from the phage is identical in nucleotide sequence to cellular SU3+ tRNATYr (7). Moreover, the synthesis of SU3+ tRNATYr from the bacteriophage 080 psu3+ is under the host's stringent control when the phage infects a stringent bacterium starved for an amino acid (8).…”

mentioning

confidence: 99%

Mutants of Escherichia coli Thermosensitive for the Synthesis of Transfer RNA

Schedl

Primàkoff

1973

186

Using a simple three-step procedure, we have isolated thermosensitive mutants of E. coli that are specifically defective in transfer RNA (tRNA) synthesis. Our procedure was designed to identify mutants that are unable to make SU3+ tRNATYr or grow at the restrictive temperature, yet under the same conditions they retain the ability to make mRNA and protein. the mature tRNA, has to be easily assayed. The tRNA gene carried by the phage 080 pSU3+ seems to meet these requirements. The infection of Escherichia coli by the phage 480 pSU3+ leads to the synthesis of tRNATYr (6). The final su3+ tRNATYr product from the phage is identical in nucleotide sequence to cellular SU3+ tRNATYr (7). Moreover, the synthesis of SU3+ tRNATYr from the bacteriophage 080 psu3+ is under the host's stringent control when the phage infects a stringent bacterium starved for an amino acid (8). This observation indicates that the promoter and the form of RNA polymerase involved in transcribing the tRNATYr gene from the phage are probably identical to those involved in transcribing tRNATYr from the bacterial chromosome. Consequently the biosynthetic steps for the synthesis of SU3+ tRNATYr from 480 pSU3+ should be identical to those used for cellular synthesis of tRNATYr and, presumably, many other tRNA species; these steps should be catalyzed by enzymes already present in uninfected cells. The phage tRNA gene meets the first two requirements. Since this gene codes for an amber suppressor, the third requirement is also satisfied. One can assay for the synthesis of this tRNA by testing for the suppression of an amber mutation in readily measurable enzymes.Our approach, therefore, was to isolate therrnosensitive (ts) cell mutants that would be unable to make functional SU3+ tRNATYr when infected with 480 psu3+ at high temperature. We expected that such mutants would also be unable to synthesize functional cellular tRNA at high temperature. Using a strain of E. coli K-12 that contains an SU3+ suppressible amber mutation in the structural gene for j3-galactosidase, we isolated cell mutants that could not synthesize 0-galactosidase at high temperature after infection with 480 pSU3+, but were able to synthesize fl-galactosidase after infection with 080 pSU3+ at low temperature. In order to be sure that these cell mutants were specifically blocked in the synthesis of functional SU3+ tRNATYr at the restrictive temperature, we tested the mutants for their ability to make j3-galactosidase after 480 plac infection. Only those cells that made 13-galactosidase after 480 plac infection at the restrictive temperature were designated as ts mutants for tRNA synthesis.The ability to make f3-galactosidase after 480 plac infection indicates that, at high temperature, the mutants are able to absorb 480, can make 13-galactosidase message, and can translate that message into protein. Thus, the inability of the mutants to suppress the amber codon in 13-galactosidase at high temperature after 480 pSU3+ infection reflects a specific 2091

“…(Lowver) Primary structure of Escherichia coli tRNATYr. The structure is drawn in a cloverleaf diagram and T1 ribonuclease oligonucleotides have been enclosed by broken lines and labeled with italic numbers (9,10). The solid lines enclose oligonucleotides shown previously to photocrosslink to bound tyrosyl-tRNA synthetase.…”

Section: Methodsmentioning

confidence: 99%

“…The two isoacceptors for E. coli tRNATyr contain 4-thiouridine at positions 8 and 9 but have no other modified bases known to be susceptible to NaBH4 (Fig. 1 Lower) (9,10). Unlike almost all other tRNAs, therefore, this species can be explored to assess the effect of reduction solely at 4-thiouridine.…”

mentioning

confidence: 99%

Covalent enzyme-RNA complex: a tRNA modification that prevents a covalent enzyme interaction also prevents aminoacylation.

Starzyk¹,

Schoemaker²,

Schimmel³

1985

Previous work indicates that aminoacyltRNA synthetases make a transient covalent adduct with cognate tRNAs, through Michael addition of an enzyme nucleophile to the carbon-6 position of uridine 8. We report the selective reduction of the 5,6 double bond of 4-thiouridine at position 8 in Escherichia coli tyrosine tRNA, so as to prevent formation of the presumed covalent enzyme-nucleic acid adduct. The completely reduced tRNA molecules are inactivated for aminoacylation. With partial reduction, a mixed pool of active and inactive molecules is created and the degree of inactivation exactly matches the extent of 4-thiouridine reduction. The active molecules recovered from this mixed pool are specifically unaltered at position 8. The results are consistent with the view that the covalent enzyme-RNA adduct is an obligatory intermediate for aminoacylation of this tRNA. Na BH4