The Ribosomal Elongation Cycle: tRNA Binding, Translocation and tRNA Release

Abstract

Experiments concerning the ribosomal elongation cycle have been performed; the following results were obtained.1. A measurement of the percentage of tightly coupled ribosomes participating in a tRNA binding reaction is obtained by determining both the stoichiometry of binding and the site location. In the case of AcPhe-tRNA it is sufficient to determine the maximal binding since at moderate Mg2+ concentration ( 5 15 mM) one ribosome cannot bind more than one AcPhe-tRNA molecule, which can be present at either the A or P site (exclusion principle).2. tRNA binding to poly(U)-programmed ribosomes does not follow the predictions of simple models with rigid tRNA binding sites as postulated by the classical A/P site model. For example, 2.7 molecules of tRNAPh' are found per ribosome at a ribosome concentration of 0.33 pM and a [14C]tRNAPh' excess over ribosomes of about 40-fold, in agreement with our previous report. At a 70s ribosome concentration of 3.3 pM and a sevenfold excess of tRNAPh' a binding of 2.5 tRNA molecules/ribosome would be expected according to the binding constants reported by us, but in fact a binding of only 1.8 molecules/70S ribosome is found. These findings explain the current discrepancies concerning the number of tRNA sites on the ribosome and confirm previous reports that three tRNA molecules can be bound simultaneously to one ribosome.3. The ternary complex EF-Tu . GTP . Phe-tRNAPhe binds to the A site at 0°C even if the P site is not occupied. Only if the P site is free does the addition of EF-G provoke a translocation to the P site at 0 "C, whereas at 37 "C the Phe-tRNAPh' 'slides' from the A to the P site without EF-G. This EF-G-independent sliding is not observed if the P site is filled by a deacylated tRNAPhc. Thus, EF-G is directly involved in the translocation reaction, and its role is not restricted to tRNA release.4. Kinetics of the puromycin reaction with Phe-tRNA and AcPhe-tRNA, respectively, were compared. The initial rate of AcPhe-puromycin formation exceeds that of Phe-puromycin formation by a factor of 3.6 (0 "C, 15 mM Mg2+).5. Poly(U)-programmed ribosomes carrying only [14C]tRNA release tRNA upon addition of EF-G. tRNA release is observed only if at least two tRNAPh" molecules are present per ribosome. The tRNA bound first is released with a higher probability than that bound second. Such a complex is not related to a classical pretranslocation complex carrying a peptidyl-tRNA at the A site. Therefore, when tRNA release during the course of an EF-G-induced translocation reaction is studied, care must bc taken that no significant amounts of the complex 70s . mRNA . (deacylated tRNA) are present.Our recent observation that E,ycherichiu coli ribosomes have, in addition to the classical A and P sites, a third tRNA binding site, the E site, which codon-specifically binds deacylated tRNA only [1,2], has revived the discussion of the number of tRNA binding sites. These findings, which were obtained with the standard nitrocellulose-filter technique, have been questioned by Gassen and...

Partial release of AcPhe‐Phe‐tRNA from ribosomes during poly(U)‐dependent poly(Phe) synthesis and the effects of chloramphenicol

“…The preparation of radioactively labeled Phe-tRNA, AcPhetRNA, Lys-tRNA and AcLys-tRNA, as well as the isolation of EF-G and EF-Tu, was according to [lo]. Unless otherwise stated, Phe-tRNA and AcPhe-tRNA were freed from deacylated tRNA by benzoylated DEAE-cellulose chromatography [2] with the modification of [5]. The degree of charging of unpurified Phe-and AcPhe-tRNA was 1000-1100 pmol/A260 unit, that of purified tRNA 1400-1500 pmol/A2,0 unit.…”

Testing an alternative model for the ribosomal peptide elongation cycle.

“…Tightly coupled 70S ribosomes were isolated from E. coli K-12 cells (strain D10) as described (1). One A20 unit of 70S ribosomes was assumed to be equivalent to 24 pmol.…”

“…Phe-tRNAPhe and Ac-PhetRNAPhe were freed from deacylated tRNA by benzoylated DEAE-cellulose chromatography (6). The preparations usually contained less than 10% deacylated tRNA.…”

“…In addition to the well-characterized aminoacyl-tRNA (A) and peptidyl-tRNA (P) sites, the Escherichia coli ribosome carries a third tRNA-binding site, the exit (E) site, which binds exclusively deacylated tRNA, depending on the codon present (1,2). The existence of this third binding site has been questioned by Schmitt et al (3), using an ultracentrifugation technique for the analysis of tRNA binding to ribosomes.…”

A highly efficient poly(U)‐dependent poly(Phe) synthesis system for the extreme halophile archaebacterium Halobacterium halobium

“…columns were as described [8]. Cells were cultivated at 37°C under continuous aeration in 100 1 medium in the presence of antifoam (Wacker Silicone SLE, Wacker-Chemie, Munich; a IO-times diluted solution was sterilized, lo-15 ml of which was added per 12 h) and harvested after 1.5 days at mid-log phase (0.15 A~60 units per ml), the generation time during log phase being 5.6-6 h. About 100 g cells per 100 1 medium were obtained, and these cells were stored at -80°C.…”