Biomedical subjects
Publications and source records attributed to M Sprinzl.
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Scanning transmission electron microscopy (STEM) was used to visualize formation of a ternary complex between the T. thermophilus elongation factor (EF) Tu.GTP and the Escherichia coli Phe-tRNA(Phe) labeled with an undecagold (Au11) cluster at minor nucleotide 3-(3-amino-3-carboxypropyl) uridine at position 47. The ternary complex was further characterized by the molecular mass and radius of gyration calculated from the mass distribution within the individual particles. Under conditions used for STEM imaging, the ternary complex is formed between Au11-labeled Phe-tRNA(Phe) and Tu.GTP in a yield up to 25%. The stoichiometry of EF-Tu.GTP to aminoacyl-tRNA (aa-tRNA) in the EF-Tu.GTP.aa-tRNA complex is 1:1, in agreement with the established view of the protein biosynthesis mechanism. The ternary complex is also formed, although to a lower extent, with GTP analogues (GMPPCP and GMPPNP, respectively), but not with Tu.GDP and nonaminoacylated tRNA(Phe) with Tu.GTP.
The elongation factor Tu (EF-Tu) encoded by the tufl gene of the extreme thermophilic bacterium Thermus thermophilus HB8 was expressed under control of the tac promoter from the recombinant plasmid pEFTu-10 in Escherichia coli. Thermophilic EF-Tu-GDP, which amounts to as much as 35% of the cellular protein content, was separated from the E coli EF-Tu-GDP by thermal denaturation at 60 degrees C. The overproduced E coli-born T thermophilus EF-Tu was characterized by: i) recognition through T thermophilus anti-EF-Tu antibodies; ii) analysis of the peptides obtained by cyanogen bromide cleavage; iii) thermostability; iv) guanine nucleotide binding activity in the absence and the presence of elongation factor Ts; and v) ternary complex formation with phenylalanyl-tRNAPhe and GTP.
The stoichiometry of the EF-Tu-GTP-aminoacyl-tRNA complex has been re-determined by a variety of methods, viz gel filtrations, fluorescence titrations, as well as hydrolysis and RNase protection experiments. The results of these experiments clearly demonstrate that one aminoacyl-tRNA interacts with only one EF-Tu-GTP molecule, in agreement with the established view and in contrast to the recently published results by Ehrenberg et al [6].
Several GTPases participate in bacterial protein biosynthesis. Initiation factor 2 controls the formation of the ribosomal initiation complex and places initiator fMet-tRNAfMet in the ribosomal P-site. Elongation factors Tu and G are responsible for codon-specific binding of the aminoacyl-tRNA to the A-site, and peptidyl-tRNA to the P-site, respectively, during the elongation phase of protein biosynthesis. Release factor 3, a GTPase which is not ubiquitous, is involved in termination and release of the nascent polypeptide. Other translation factors, including initiation factors 1 and 3, elongation factor Ts, release factors 1 and 2, and ribosomal release factor do not belong to the family of GTP/GDP binding proteins. The guanosine nucleotide binding domains of the GTPases involved in translation are structurally related to the Galpha subunit of heterotrimeric G proteins and to the proteins of the Ras family. We have identified and sequenced all genes coding for translation factors in the extreme thermophile Thermus thermophilus. The proteins were overproduced in Escherichia coli, purified, biochemically characterised and used for crystallisation and structural analysis. Further biochemical investigations were aimed at gaining insight into the molecular mechanism underlying the regulation of the GTPase activity of the translation factors, and to elucidate the role of their ribosomal binding sites in this process.