City Research Online

Abstract

1. Elongation factor - Tu (EF-Tu) occurs in large quantities in bacterial cells: as much as 10% in wild-type E.coli, reducing to 4% in a slow-growing mutant strain, E.coli 02216, with a kirromycin-resistant EF-Tu. Isoelectric focussing suggests that of the two E.coli genes for EF-Tu, only one may be functioning in E.coli 02216. Evidence is presented which supports a membrane-association for some of the EF-Tu in the cell.

2. EF-Tu aids the tRNA-mediated translation of mRNA into proteins, through formation of the ternary complex EF-Tu.GTP.aminoacy1-tRNA, which binds to the ribosome.mRNA complex. There, after codon-anticodon interaction, GTP is hydrolyzed and EF-Tu.GDP leaves the ribosome to be recycled. The antibiotic kirromycin binds to EF-Tu in a 1:1 ratio inducing a similar GTPase activity in the elongation factor, which can be further stimulated by ribosomes and/or aminoacyl-tRNA.

3. In the absence of ribosomes and aminoacyl-tRNA, and above 400m, monovalent cations stimulate the EF-Tu.kirromycin GTPase activity in the Li*>Na">k*S>NH,">Cs"SmMe,N", concomitantly changing the Km for GTP in this reaction. Below 400mM [n*] ribosomes and aminoacyltRNA stimulate this GTPase activity with a monovalent cation specificity: NH, ">> K*>Li" = Cs">Na‘>Me,N", without influencing the kK, for GTP. Ribosomes can completely replace the absolute requirement for monovalent cations in stimulating the EF-Tu.kirromycin GTPase activity. In the physiological EF-Tu GTPase, small monovalent cations (Na* and La’) also stimulate the GTP hydrolysis at 30mM | » and concomitantly change the Kn for the reaction.

4. Free divalent cations are not absolutely required for EF-Tu. kirromycin GTPase activity, and at 200mM [n*] only stimulate in the additional presence of ribosomes. In the physiological EF-Tu GTPase increasing divalent cation concentration removes the requirement for mRNA of this reaction. For the kirromycin-induced EF-Tu GTPase activity there is a marked interdependence between pH and monovalent cation concentration in the absence of free Me?" supporting the location of a highly charged anionic region close to the catalytic centre for GTP
hydrolysis on EF-Tu.

5. EF-Tu interacts with the ribosome largely via the 50S subunit. Although proteins L7/L12 are involved in this interaction, they are dispensible for the expression of the EF-Tu.kirromycin GTPase in the presence of 30S subunits and aminoacyl-tRNA, together with a large number of other ribosomal proteins. These results suggest a complex interaction between EF-Tu and the ribosome, which is supported by other experiments using the antibiotic thiostrepton.

6. The results are discussed in relation to the physiglogical role of EF=Tu and it is considered that the elongation factor is critically involved in the recognition mechanism whereby miscoding is .avoided.

Publication Type:	Thesis (Doctoral)
Subjects:	Q Science Q Science > QD Chemistry
Departments:	School of Science & Technology School of Science & Technology > School of Science & Technology Doctoral Theses Doctoral Theses

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