Unwinding single RNA molecules using helicases involved in eukaryotic translation initiation.
The small (40 S) subunit of the eukaryotic ribosome may have to scan more than 2000 nucleotides (>600 nm) from its 5'cap recruiting point on an mRNA molecule before initiating on a translation start codon. As with many other processes in living cells, including transcription, editing, mRNA splicing, pre-rRNA processing, RNA transport and RNA decay, scanning is facilitated by helicase activity. However, precise quantitative data on the molecular mechanism of scanning, including the roles of helicases, are lacking. Here, we describe a novel atomic force microscopy (AFM)-based procedure to examine the roles of two yeast helicases, eIF4A and Ded1, previously implicated in translation initiation by genetic and biochemical studies. Our results show that eIF4A, especially in the presence of its "cofactor" eIF4B, promotes ATP-dependent unwinding of localised secondary structure in long RNA molecules under tensional loading, albeit only at high protein:RNA ratios. Thus eIF4A can act to separate only a limited number of base-pairs, possibly via a steric unwinding mechanism. In contrast, Ded1 is more effective in reducing (by up to 50 pN at an AFM loading rate of 14 nNs(-1)) the force necessary to disrupt an RNA stem-loop, and thus shows significant kinetic competence to facilitate fast unwinding. These single molecule experiments indicate that Ded1 is likely to act as the more potent unwinding factor on natural mRNA substrates.
Marsden
Steven
S
Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester M1 7ND, UK.
Nardelli
Maria
M
Linder
Patrick
P
McCarthy
John E G
JE
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D053487:DEAD-box RNA Helicases; D039601:Eukaryotic Initiation Factor-4A / Q000737:chemistry / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors / Q000737:chemistry / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D020365:RNA Helicases / Q000737:chemistry / Q000378:metabolism*; D012331:RNA, Fungal / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger; D012270:Ribosomes; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*
D018797:Cell Cycle Proteins; D039642:Eukaryotic Initiation Factors; D010448:Peptide Initiation Factors; D012331:RNA, Fungal; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; C031799:eIF-4B; D000255:Adenosine Triphosphate; D039601:Eukaryotic Initiation Factor-4A; D000251:Adenosine Triphosphatases; C492152:DED1 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases