Dynamics and processivity of 40S ribosome scanning on mRNA in yeast

• Published in: Molecular microbiology Vol. 51; no. 4; pp. 987 - 1001
• Main Authors: Berthelot, Karine, Muldoon, Mark, Rajkowitsch, Lukas, Hughes, John, McCarthy, John E. G.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.02.2004; Blackwell Science; Blackwell Publishing Ltd
• Subjects: 5' Untranslated Regions - physiology; Biological and medical sciences; Cell Cycle Proteins - physiology; Codon, Initiator - metabolism; DEAD-box RNA Helicases; Eukaryotic Initiation Factor-4A - physiology; Eukaryotic Initiation Factors - physiology; Fundamental and applied biological sciences. Psychology; Fungal Proteins - physiology; Microbiology; Peptide Chain Initiation, Translational; Protein Biosynthesis; ribosome 40S; Ribosomes - physiology; RNA Caps - metabolism; RNA Helicases - physiology; RNA, Bacterial - metabolism; RNA, Messenger - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - physiology; Yeast; Messenger RNA; Microbiology; 40S-Ribosome
• DOI: 10.1046/j.1365-2958.2003.03898.x
• ISSN: 0950-382X

Summary The eukaryotic 40S ribosomal subunit locates the translation initiation codon on an mRNA via the so‐called 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′→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.