Pseudouridine synthases are enzymes that catalyze the isomerization of uridine to pseudouridine (Ψ) in RNA. The other common modification of RNA is the 2'-O-ribose methylation. The pseudouridine synthases from the three domains of life namely, bacterial TruB, archaeal Pus10 and eukaryal Pus4, are responsible for the formation of the universally conserved Ψ55 in the T-arm of tRNAs. We have carried out a comparative analysis of their activities on two different tRNA substrates namely tRNATrp and tRNAMet. Our findings support the observation that TruB absolutely requires the U54:A58 reverse Hoogsteen base pairing when it encounters the minimal T-arm-substrate. However, this requirement is relaxed when the length of the tRNA substrate is increased. Pus10 does not require this reverse Hoogsteen pair for its activity when tested in lower salt. However, as the salt concentration is increased, the Ψ formation is lost in the minimal substrate lacking this base pairing. Pus4, on the other hand, can modify the T-arm substrate and does not have an absolute requirement for the U54:A58 pairing. The Ψ54 and Ψ55 activity of Pus10 and TruB was also compared and it was seen that as the length of the substrate increased, the Pus10 activity became more specific for Ψ54 formation while TruB did not show any Ψ54 formation as reported earlier. Additionally, the effect of Cm56 methylation on the activities of TruB, Pus10 and Pus4 was determined in vitro. TruB shows a decrease in Ψ55 formation in the C56 methylated tRNAMet transcript but does not seem to affect the C56 methylated tRNATrp transcript. Pus10 and Pus4, on the other hand show a lower Ψ55 formation in both the C56 methylated transcripts. In vivo analysis of the effect of Cm56 methylation on the activity of TruB in E. coli was not conclusive and requires further study.

No description available.

The <i>truB</i> gene of <i>Escherichia coli </i>encodes the pseudouridine (ψ)-55 synthase and is responsible for modifying all tRNA molecules in the cell at the U55 position. The aim of this project was to investigate the physiological role of the truB gene and ψ55 tRNA modification. A <i>truB</i> null mutant created in this work, grew normally on all growth media tested, but exhibited a competitive disadvantage in extended co-culture with its wild-type progenitor. The mutant phenotype could be complemented by both the cloned <i>truB</i> gene and by a D48C, catalytically-inactive, allele of <i>truB. </i> The proteome of the <i>truB</i> mutant also contained altered levels of intermediates involved in biogenesis of the outer membrane proteins (OMP), OmpA and OmpX. In addition, the <i>truB</i> mutation reduced the basal expression from two s^E promoters, <i>degP </i>and <i>rpoH</i>P3 although this could not be complemented with cloned <i>truB</i>. The <i>truB</i> mutant also exhibited a defect in survival of rapid transfer from 37^oC to 50^oC. This mutant phenotype could be complemented by the cloned <i>truB</i> gene but not by a D48C, catalytically-inactive, allele of <i>truB</i>. The temperature-sensitivity of <i>truB</i> mutants could be enhanced by combination with a mutation in the <i>trmA</i> gene, encoding the enzyme modifying the universal U54 tRNA nucleoside, but not by mutations in <i>trmH</i> , encoding the enzyme catalysing the formation of Gm18. Three novel aspects to the phenotype of <i>truB </i>mutants have been identified. Importantly the data support the hypothesis that TruB-effected ψ55 modification of tRNA is not essential but contributes to thermal stress tolerance in <i>E. coli</i>, possibly by optimising the stability of the tRNA population at high temperatures.

Akademisk avhandling--Gothenburg. / Extra t.p., with thesis statement, inserted. Bibliography: p. 23-24.

Akademisk avhandling--Gothenburg. / Extra t.p., with thesis statement, inserted. Bibliography: p. 23-24.

The aminoacyl-tRNA synthetases (aaRSs) are the universal set of enzymes responsible for attaching amino acids to tRNA to be used as substrates in the process of protein translation. As these enzymes act at the transition between nucleic acids and proteins, their specificity of action is critical for maintaining the fidelity of the genetic code. From a mechanistic standpoint, aaRS specificity is enforced by a complex series of tRNA structural and chemical elements that collectively make up its identity set and serve to distinguish one tRNA from another. Based on sequence, structure, and oligomeric differences, the aaRS family has been partitioned into two classes, each of which is responsible for roughly half of the 22 genetically encoded amino acids. In the studies presented here, pre-steady-state kinetic methods were employed to measure individual events that collectively make up the catalytic cycle of the class II Escherichia coli Histidyl-tRNA Synthetase (HisRS) in order to elucidate the nature of its enzymatic activity and determine how these events contribute to the exquisite specificity between enzyme and tRNA. The results presented here indicate indentiy elements of tRNAHis regulate the activity of the amino acid activation and aminoacyl transfer half reactions. Additional evidence suggests communication between active sites of the HisRS homodimer plays a role in establishing an alternating cycle of catalysis in the steady state.

No description available.

Direct synthesis of aminoacyl-tRNA occurs using α-N-tBoc-protected aminoacyl phosphates and lanthanum salts. Deprotection of aminoacyl tRNA is essential prior to translation; however, the conditions of tBoc deprotection causes tRNA degradation. It was found that α-N-pentenoyl-protected aminoacyl phosphates, deprotected under mild conditions, are effectively used in lanthanum-mediated acylation of tRNA analogs. This provides an alternative route for aminoacyl-tRNA synthesis that maintains tRNA structure. Also, it was determined that α-N-deprotection of aminoacyl phosphates prior to aminoacylation still produces aminoacylated tRNA analogs. This establishes that acyl phosphates activate amino acids without inducing self-condensation, presumably due to electrostatic repulsion. Direct quantification of lanthanum-mediated tRNA aminoacylation was additionally undertaken utilizing a radiolabelled tRNA assay. From this, it was shown that lanthanum-mediated acylation does not promote deacylation and degradation of tRNA. These results have provided insight into lanthanum-mediated acylation of tRNA, ultimately allowing for use of the reagent in ribosomal translation.

Direct synthesis of aminoacyl-tRNA occurs using α-N-tBoc-protected aminoacyl phosphates and lanthanum salts. Deprotection of aminoacyl tRNA is essential prior to translation; however, the conditions of tBoc deprotection causes tRNA degradation. It was found that α-N-pentenoyl-protected aminoacyl phosphates, deprotected under mild conditions, are effectively used in lanthanum-mediated acylation of tRNA analogs. This provides an alternative route for aminoacyl-tRNA synthesis that maintains tRNA structure. Also, it was determined that α-N-deprotection of aminoacyl phosphates prior to aminoacylation still produces aminoacylated tRNA analogs. This establishes that acyl phosphates activate amino acids without inducing self-condensation, presumably due to electrostatic repulsion. Direct quantification of lanthanum-mediated tRNA aminoacylation was additionally undertaken utilizing a radiolabelled tRNA assay. From this, it was shown that lanthanum-mediated acylation does not promote deacylation and degradation of tRNA. These results have provided insight into lanthanum-mediated acylation of tRNA, ultimately allowing for use of the reagent in ribosomal translation.

No description available.