Differential effects of nucleotide analogs on scanning-dependent initiation and elongation of mammalian mRNA translation in vitro
Codon-anticodon interactions are central to both the initiation and elongation phases of eukaryotic mRNA translation. The obvious difference is that the interaction takes place in the ribosomal A-site during elongation, whereas the 40S ribosomal subunit and associated initiation factors scan the mRN...
Saved in:
Published in | RNA (Cambridge) Vol. 16; no. 6; pp. 1130 - 1137 |
---|---|
Main Authors | , |
Format | Journal Article |
Language | English |
Published |
United States
Cold Spring Harbor Laboratory Press
01.06.2010
|
Subjects | |
Online Access | Get full text |
Cover
Loading…
Summary: | Codon-anticodon interactions are central to both the initiation and elongation phases of eukaryotic mRNA translation. The obvious difference is that the interaction takes place in the ribosomal A-site during elongation, whereas the 40S ribosomal subunit and associated initiation factors scan the mRNA sequence in search of an initiation codon with Met-tRNA(i) bound in the P-site, ceasing once codon-anticodon interaction is established at the AUG. As an indirect test of whether the two mechanisms of mRNA sequence inspection are basically similar or not, the effects of six different uridine analog substitutions in the mRNA were examined in reticulocyte lysate translation assays and 80S initiation complex formation assays. Four constructs, each with the same reporter coding sequence, were used, differing in whether the initiation codon was AUG or ACG, and in whether the 5'-UTR had U residues or not. Three analogs (5-bromoU, 5-aminoallylU, and pseudoU) inhibited both elongation and initiation, but the other three had striking differential effects. Ribothymidine had a negligible effect on elongation but caused a approximately 50% inhibition of initiation, with little effect on actual AUG recognition, which implies that inhibition must have occurred at some earlier step in initiation. In complete contrast, 2' deoxyU was prohibitive to elongation but had no effect on initiation, and 4-thioU actually stimulated initiation but quite strongly inhibited elongation processivity. These results show that the detailed mechanisms of inspection of the mRNA sequence during scanning-dependent initiation and elongation must be considerably different. |
---|---|
Bibliography: | Present address: Department of Molecular and Cell Biology and Center of Integrative Genomics, University of California at Berkeley, Berkeley, CA 94720-3204, USA. Reprint requests to: Richard J. Jackson, Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, United Kingdom; e-mail: rjj@mole.bio.cam.ac.uk; fax: 44-1223-766002. |
ISSN: | 1355-8382 1469-9001 |
DOI: | 10.1261/rna.1978610 |