Datensatz

Zusammenfassung

In this work the phosphorothioate technique is applied that allows a precise assessment of the accessibility of each individual phosphate group within RNA. The measurements were preformed with deacylated tRNAs bound to the ribosomal P site under various buffer conditions, and with a tRNA in acylated and deacylated form present at the ribosome in various functional states. The buffer conditions used have led in the past to different results concerning the tRNA location with important implications to elongation cycle models and the mechanism of translocation. The results have revealed that (i) the tRNA undergoes significant conformational changes already in solution, if physiological conditions at low Mg2+ and polyamines where compared with conventional buffer conditions at high Mg2+ in the absence of polyamines- Surprisingly, the tRNA seems to adopt a more compact structure under low Mg2+ conditions plus polyamines as compared to high Mg2+ (10 and 20 mM, conventional system). (ii) If bound to the programmed P site the tRNAs seem to be forced into the same structure irrespective of the buffer system used, since tRNAs under all conditions tested demonstrate an almost identical accessibility pattern. The very same accessibility pattern under various buffer conditions indicates the same micro-environment of the tRNAs, a result that notably contrasts the significant differences in tRNA location cryo-EM has revealed under corresponding conditions. Taken together, these results represent a further support for a ribosomal carrier that binds tightly the tRNAs and moves them during translocation from the PRE to the POST state. Another experiment compared the protection pattern of a tRNA at the programmed and non-programmed P site of 70S ribosomes (i.e. in the presence or absence of mRNA). The surprising result was that in the non-programmed 70S ribosome the accessibility pattern those caused by 30S contacts were practically absent, whereas the 50S part of the accessibility pattern was almost normal. Obviously, codon-anticodon interactions at the P site are causing the 30S contacts even outside the anticodon demonstrating an influence of this interaction on the 30S-tRNA contacts in general. 2In the second part the phosphorothioate results are compared with the crystal structure of Thermus thermophilus 70S ribosomes carrying three tRNAs. An excellent agreement is found concerning the results with accessibility patterns derived from tRNAs bound to single subunits and the tRNA contacts with both subunits within the P site of 70S ribosomes. A new and significant insight could be obtained concerning the tRNA fixation points: Not only the tRNA nucleotides important for fixation of the tRNA at the P site are universally conserved as noted earlier, but also the nucleotides of the rRNA and ribosomal proteins involved in tRNA fixation are universally or highly conserved. Finally, a compilation of the available facts from cryo-EM, crystallographic studies of functional complexes and biochemical data do not favor the hybrid-site model, but are consistent with the α-ε model.