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The overall aim of this work was to gain further insight into the biochemistry of the mechanism of mammalian 40S ribosomal scanning during initiation of mRNA translation. One project was designed to measure the speed of scanning over different lengths of 5’-untranslated region (5’-UTR). To this end, two related mRNAs were co-translated at room temperature (26°C) in vitro; they differed only in that one had a small deletion in the coding region, and one had a longer 5’-UTR than the other. The time-lags between addition of the mRNA to the assay and the first initiation events on the two mRNAs were measured, and the difference in the two time-lags was taken as the time required to scan the additional 5’-UTR length. By this method scanning speeds of 4.7 nt/second and 7.7 nt/second were observed in two different batches of lysates for a 5’-UTR based on that of influenza virus (A strain) NS1 mRNA. With a range of different lengths obtained by serial repetition of the tobacco mosaic virus 5’-UTR, the measured scanning speeds ranged from 1.8 to 8.0 nt/second. Surprisingly, the apparent speed increased with increasing 5’-UTR length, suggesting that the 40S subunits may gather speed during scanning, or, alternatively, may have an increasing tendency to by-pass segments of the 5’-UTR as its length is increased. The nature of the interactions between the 40S ribosomal subunit and the mRNA during scanning was investigated by substituting modified nucleotides (mainly modified U residues). The four mRNAs examined all had the same coding region containing numerous U residues; two had an AUG start codon and two an ACG, while two had a 5’-UTR lacking any U residues. These mRNAs were tested in translation assays, and also in 80S initiation complex formation assays. Substitution with 5-bromoU, 5-aminoallyIU or pseudoU had negative effects both on elongation (seen when the modification was present only in the coding region), and on initiation. However, substitution by other modified U residues affected either elongation or initiation, but not both. Ribothymidine (5-methylU) substitution had a negligible effect on elongation, but, most surprisingly, its presence in the 5’-UTR reduced initiation by over 50, irrespective of the nature of the start codon, suggesting that it is inhibitory to 40S subunit loading or scanning. |
