The Interaction of Elongation Factor G with N -Acetylphenylalanyl Transfer RNA·Ribosme Complexes

Abstract

N-Acetyl-Plie-tRNA, nonenzymically bound to the acceptor site of Escherichia coli ribosomes, readily undergoes translocation in the presence of elongation factor (EF)-G and GTP. The translocated N-acetylPhe-tRNA, bound to the ribosomal donor site, prevents further interaction of EF-G with the ribosome, for it inhibits the GTP hydrolysis that takes place in the presence of EF-G and ribosomes and it decreases the formation of either the GDP EF-G fusidic acid ribosome complex or the 5'-guanylylmethylenediphosphonate-EF-Gribosome complex. Deacylation with puromycin of the donor site-bound NY-acetyl-Phe-tRNA reverses these inhibitions, even though the tRNAPhe moiety remains bound to the ribosome. These results suggest that ribosomes complexed with messenger RNA and peptidyvl-tRNA may be restricted in their ability to interact with EF-G to that part of the elongation cycle when peptidyl-tRNA is in the acceptor site, and deacylated tRNA in the donor site.

The Binding of Tritiated Elongation Factors 1 and 2 to Ribosomes from Krebs II Mouse Ascites Tumor Cells

“…The strong preference exhibited by A-site and P-site ribosomes for the binding of EF-2 and EF-1 respectively is in line with previous observations made in this laboratory [3] and with results presented by Nombela and Ochoa for ribosomes from artemia salina [6] and by Modolell e t a f . for bacterial ribosomes [19]. Taken together with the fact that in the presence of GTP, the interaction of both factors with the ribosome is a very transient one, the results imply that a mutual interference of the two elongation factors with respect to their binding and function does not occur under conditions in vivo, as was also concluded by Baliga et af.…”

The Interaction of Fusidic Acid with Peptidyl‐Transfer‐Ribonucleic‐Acid · Ribosome Complexes

“…I n this cxperimcnt i n c u b a h n wii\ at 30 polypeptide synthesis. This hydrolysis was probably due to free ribosomes present in the reaction mixturc, since ribosomes complexed with Ac-Phc-tRNA and polysoinal ribosomes are impaired in complementing EF-G in the uncoupled GTP hydrolysis [20,22]. This interpretation is supported by the observation that the scnsitivity to fusidic acid of the GTP hydrolysis occurring in mixtures containing only free ribosomes closely parallelled that of the complete reaction mixtures (Fig.…”

Translocation in ribosomes by attachment—detachment of elongation factor G without GTP cleavage: Evidence from a column‐bound ribosome system

“…If the experiments of both Vazquez et al and ours are taken to be correct, then the difference in the results can be explained only by the difference in experimental conditions: competence to puromycin does not appear when EF-G with GMPPCP are still present in the medium and in the ribosomes [3], but it appears when EF-G with GMPPCP is washed off ( fig.1 and table 1). From this it follows that; 1) either the attachment of EF-G with GMPPCP to the ribosome is sufficient to perform translocation, but the bound EF-G inhibits the puromycin reaction; or 2) translocation is performed (completed) by the detachment of EF-G.…”

“…In their experiments Vazquez's group [3] did not succeed in producing translocation by substitution of GMPPCP for GTP. If the experiments of both Vazquez et al and ours are taken to be correct, then the difference in the results can be explained only by the difference in experimental conditions: competence to puromycin does not appear when EF-G with GMPPCP are still present in the medium and in the ribosomes [3], but it appears when EF-G with GMPPCP is washed off ( fig.1 and table 1).…”