SummaryThe eukaryotic 40S ribosomal subunit locates the translation initiation codon on an mRNA via the socalled scanning process that follows 40S binding to the capped 5 ¢ ¢ ¢ ¢ end. This key step in translation is required for the expression of almost all eukaryotic genes, yet the mechanism and dynamics of scanning are unknown. We have performed quantitative studies in vivo and in vitro of the movement of yeast 40S ribosomes along 5 ¢ ¢ ¢ ¢ untranslated regions (UTRs) of different lengths. 40S subunits perform cap-dependent scanning with high processivity for more than 1700 nucleotides in cells of Saccharomyces cerevisiae . Moreover, the observed rates of expression indicate that scanning is performed by an untethered 40S subunit that has been released from the 5 ¢ ¢ ¢ ¢ cap complex. Unexpectedly, the capability to maintain scanning competence on a long 5 ¢ ¢ ¢ ¢ UTR is more dependent on the Ded1/Dbp1 type of helicase than on eIF4A or eIF4B. In a yeast cell-free extract, scanning shows reduced processivity, with an estimated net 5 ¢ ¢ ¢ ¢AE AE AE AE 3 ¢ ¢ ¢ ¢ rate of approximately 10 nucleotides per second at 26 ∞ ∞ ∞ ∞ C. We have developed a biased bidirectional walking model of ribosomal scanning that provides a framework for understanding the above observations as well as other known quantitative and qualitative features of this process.

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SummaryWe describe post-transcriptional gene regulation in yeast based on direct RNA-ligand interaction. Tetracycline-dependent translational regulation could be imposed via specific aptamers inserted at two different positions in the 5 ¢ ¢ ¢ ¢ untranslated region (5 ¢ ¢ ¢ ¢ UTR). Translation in vivo was suppressed up to ninefold upon addition of tetracycline. Repression via an aptamer located near the start codon (cap-distal) in the 5 ¢ ¢ ¢ ¢ UTR was more effective than repression via a capproximal position. On the other hand, suppression in a cell-free system reached maximally 50-fold and was most effective via a cap-proximal aptamer. Examination of the kinetics of tetracycline-dependent translational inhibition in vitro revealed that preincubation of tetracycline and mRNA before starting translation led not only to the fastest onset of inhibition but also the most effective repression. The differences between the behaviour of the regulatory system in vivo and in vitro are likely to be related to distinct properties of mRNP structure and mRNA accessibility in intact cells as opposed to cell-extracts. Tetracycline-dependent regulation was also observed after insertion of an uORF sequence upstream of the aptamer, indicating that our system also targets reinitiating ribosomes. Polysomal gradient analyses provided insight into the mechanism of regulation. Cap-proximal insertion inhibits binding of the 43S complex to the cap structure whereas start-codon-proximal aptamers interfere with formation of the 80S ribosome, probably by blocking the scanning preinitiation complex.