Beyond the Identification of Transcribed Sequences:
Functional, Evolutionary and Expression Analysis
12th International Workshop
October 25-28, 2002
Washington, DC

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Global Analysis of Translation in Yeast

Yoav Arava1, Yulei Wang1, John D. Storey2, Patrick O. Brown1,3 and Daniel Herschlag1
Departments of Biochemistry 1, Statistics 2 and HHMI 3, Stanford University

We have performed a global analysis of translation in yeast to better understand the global gene expression program and to obtain new insights into translation mechanisms. Quantitative microarray analysis of mRNAs from fractions across sucrose gradients has allowed us to assemble a database describing ribosome association for thousands of genes.

Mining the ribosomes association database revealed that, for nearly all genes, the majority of mRNA molecules are associated with ribosomes and presumably engaged in translation, indicative of a very efficient recruitment of newly synthesized molecules into the translating pool. The number of ribosomes associated with mRNAs increases with increasing open reading frame (ORF) length, consistent with longer transit times for ribosomes translating longer ORFs. Surprisingly however, the density of ribosomes (i.e., the number of ribosomes per unit ORF length) decreases with increasing ORF length, suggesting lower translation efficiency for longer ORFs. Possible origins for such an effect are slower initiation rates for longer mRNAs, incomplete processivity during translocation steps (i.e., ribosomes falling off of the mRNA during elongation) and slow termination rates. These models yield physically distinct predictions: an even distribution of ribosomes along the mRNA is expected in the model of slower initiation, lower density of ribosomes towards the 3 end of the mRNA is expected if ribosomes dissociate during translocation, and higher density at the 3 end will be observed if termination is slow.

To determine the ribosome density in different regions of a particular mRNA, we developed a system that involves specific cleavage using antisense oligonucleotides followed by separation on a sucrose gradient. We used it to compare the number of ribosomes on the 5 and 3 halves for several mRNAs. In each case both halves have a similar number of ribosomes. These results strongly suggest that elongation is highly processive and that termination is not generally rate-limiting. We propose that longer mRNAs have lower translation efficiency due to slower initiation rates, perhaps the result of a more distal 3 polyA tail.

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