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S Z Wahab

Publications and source records attributed to S Z Wahab.

8 recordsLinked to original sources
PubMed1993

Effects of tRNA(1Leu) overproduction in Escherichia coli.

Strains of Escherichia coli have been produced which express very high levels of the tRNA(1Leu) isoacceptor. This was accomplished by transforming cells with plasmids containing the leuV operon which encodes three copies of the tRNA(1Leu) gene. Most transformants grew very slowly and exhibited a 15-fold increase in cellular concentrations of tRNA(1Leu). As a result, total cellular tRNA concentration was approximately doubled and 56% of the total was tRNA(1Leu). We examined a number of parameters which might be expected to be affected by imbalances in tRNA concentration: in vivo tRNA charging levels, misreading, ribosome step time, and tRNA modification. Surprisingly, no increase in intracellular ppGpp levels was detected even though only about 40% of total leucyl tRNA was found to be charged in vivo. Gross ribosomal misreading was not detected, and it was shown that ribosomal step times were reduced between two- and threefold. Analyses of leucyl tRNA isolated from these slow-growing strains showed that at least 90% of the detectable tRNA(1Leu) was hypomodified as judged by altered mobility on RPC-5 reverse-phase columns, and by specific modification assays using tRNA(m1G)-methyltransferase and pseudo-uridylate synthetase. Analysis of fast-growing revertants demonstrated that tRNA concentration per se may not explain growth inhibition because selected revertants which grew at wild-type growth rates displayed levels of tRNA comparable to that of control strains bearing the leuV operon. A synthetic tRNA(1Leu) operon under the control of the T7 promoter was prepared which, when induced, produced six- to sevenfold increases in tRNA(1Leu) levels. This level of tRNA(1Leu) titrated the modification system as judged by RPC-5 column chromatography. Overall, our results suggest that hypomodified tRNA may explain, in part, the observed effects on growth, and that the protein-synthesizing system can tolerate an enormous increase in the concentration of a single tRNA.

Bacterial Proteins↗
PubMed1992

Structural requirements for tRNA methylation. Action of Escherichia coli tRNA(guanosine-1)methyltransferase on tRNA(1Leu) structural variants.

The Escherichia coli enzyme tRNA(m1G)methyltransferase, one of a group of post-transcription tRNA-modifying enzymes, shows remarkable specificity in selecting the tRNA species and the specific guanosine base to be methylated. To examine the structural basis of this specificity, we synthesized a total of 15 modifications of tRNA(1Leu) and measured their methylation reaction kinetics in vitro. Elimination of any one of the three tRNA side loops, the V loop, the T loop, or the D loop, reduced the Vmax for methylation by about 1 order of magnitude. Elimination of all three side loops reduced Vmax by about 2 orders of magnitude. Clearly, gross tRNA structure is important for full enzyme activity. At the bottom of the stem proximal to the anticodon loop, in the pair at positions 31-39, substitution of a G-C for a C-G, a change that should not weaken the helical structure, had little effect on Vmax or Km. However, substitution of a G for a C increased Vmax and Km, whereas substitution of a C for G sharply reduced Vmax and, to a lesser extent, Km. These results appear to be a consequence of the principle that purines are better than pyrimidines in the stacking of adjacent bases for stability. Stacking in the stem structure appears to be important for methylation enzyme activity. In the anticodon loop itself, changing a U to a C had little effect, but changing the G of the anticodon to a C reduced Vmax over 20-fold, demonstrating the importance of the presence of the anticodon G adjacent to the G being methylated for enzyme recognition.

Anticodon↗
PubMed1989

Flanking sequences are required for efficient transcription and stable complex formation for the human tRNAiMet3-coding gene.

An analysis of 5' and 3' deletions of the human tRNAiMet3 gene has revealed upstream regions required for efficient transcription and stable complex formation in vitro. The 5' boundary of this essential region lies between nucleotides -39 to -18 (start point = + 1), and it has been shown that 3'-flanking sequences near the first termination site are also important for stable complex formation. The transcriptional efficiency of two non-allelic loci (TMET3 and TMET2) has been compared and TMET2 is more active. An analysis of chimeric (hybrid) genes indicates that much of the difference seen is due to 5'-flanking sequences and that there may be complex interactions between 5' and 3' sequences.

Animals↗
PubMed1987

A human tRNA(iMet) gene produces multiple transcripts.

A third nonallelic locus of the human methionyl-tRNA multigene family (tRNA(iMet-3) was isolated. This gene, unlike two other tRNA(iMet) loci, lacks a remarkable run of T and C residues which functions as a termination of transcription signal. Instead, three tandem termination signals, each containing no more than four thymidylate residues, function as relatively inefficient termination signals. As a result, polymerase readthrough generates at least three transcripts in vitro. The efficiency of apparent termination varies significantly at these sites. All resulting transcripts appear to be processed in vitro.

Base Sequence↗
PubMed1986

Comparison of the enzymatic behavior of high molecular weight and free lysyl-tRNA synthetase from rat liver: kinetic analysis of lysylation of tRNA.

Lysyl-tRNA synthetase occurs in the high molecular weight form in rat liver. The high molecular weight lysyl-tRNA synthetase has been previously demonstrated to exist as multienzyme complexes of aminoacyl-tRNA synthetases. The multienzyme complexes can be dissociated by hydrophobic interaction chromatography and yield fully active, free lysyl-tRNA synthetase. The free form is found to be twice as active as the complexed form in lysylation. Bisubstrate and product inhibition kinetics of lysylation are systematically carried out for highly purified free lysyl-tRNA synthetase and the 18 S synthetase complex. Surprisingly, the two enzyme forms exhibit distinctly different kinetic patterns in bisubstrate and product inhibition kinetics under identical conditions. The 18 S synthetase complex shows kinetic patterns consistent with an ordered bi uni uni bi ping pong mechanism, while the results of free lysyl-tRNA synthetase do not. We conclude that structural organization of lysyl-tRNA synthetase beyond quaternary structure of proteins may alter the enzyme behavior.

Amino Acyl-tRNA Synthetases↗
PubMed1985

Influence of supramolecular structure on the enzyme mechanisms of rat liver lysyl-tRNA synthetase-catalyzed reactions. Synthesis of P1,P4-bis(5'-adenosyl)tetraphosphate.

Lysyl-tRNA synthetase, dissociated from the multienzyme complexes of aminoacyl-tRNA synthetases from rat liver, was previously found to be 6-fold more active than the synthetase complex in the enzymatic synthesis of P1,P4-bis(5'-adenosyl)tetraphosphate. The bi-substrate and product inhibition kinetics of the reaction are analyzed. Free lysyl-tRNA synthetase exhibits distinctly different kinetic patterns from those of an 18 S synthetase complex containing lysyl-tRNA synthetase. The 18 S synthetase complex shows kinetic patterns which are consistent with an ordered Bi Uni Uni Bi ping-pong mechanism. Free lysyl-tRNA synthetase shows kinetic patterns consistent with a random mechanism. The differences in the enzymatic properties are attributed to the organization of the supramolecular structure of the synthetase complex. The results suggest that association of the synthetases may affect the mechanisms of the synthesis of AppppA.

Adenine Nucleotides↗
PubMed1985

Synthesis of diadenosine 5',5''' -P1,P4-tetraphosphate by lysyl-tRNA synthetase and a multienzyme complex of aminoacyl-tRNA synthetases from rat liver.

An 18 S multienzyme complex of aminoacyl-tRNA synthetases is found to be active in the synthesis of diadenosine-5',5'''-P1,P4-tetraphosphate (AppppA). Most of the activity is attributed to lysyl-tRNA synthetase in the complex. Free lysyl-tRNA synthetase dissociated from the synthetase complex is about 6-fold more active than the complex in AppppA synthesis, while their apparent Michaelis constants for ATP and lysine are similar. AMP, which reportedly activates AppppA synthesis (Hilderman, R.H. (1983) Biochemistry 22, 4353-4357), has no effect on AppppA synthesis. The higher activity of free Lys-tRNA synthetase is in part due to the higher stimulation of AppppA synthesis by Zn2+. These results suggest that association of aminoacyl-tRNA synthetases may affect AppppA synthesis.

Adenine Nucleotides↗