Biosynthesis of reovirus-specified polypeptides. Analysis of ribosome pausing during translation of reovirus S1 and S4 mRNAs in virus-infected and vector-transfected cells.
The steady-state distribution of translating ribosomes on the reovirus s1 and s4 mRNAs was determined using a sensitive primer extension inhibition assay and cDNA clones of the reovirus S1 and S4 genes. Positions of significant ribosome pausing included the sites of initiation and termination of translation, both in vivo in reovirus-infected mouse fibroblast L cells and in vitro in rabbit reticulocyte lysates. The patterns of ribosome pausing detected in vivo and in vitro were comparable. Ribosomes were far less uniformly distributed along the polycistronic s1 mRNA than they were along the monocistronic s4 mRNA. During reovirus infection, ribosome pausing was detected at the initiation codons of the s1 mRNA sigma 1 and sigma 1ns open reading frames and the s4 mRNA sigma 3 open reading frame, direct evidence that initiation is a slow step of translation in vivo. Quantitative analysis of reovirus mRNA translation in infected and transfected cells suggested that ribosomes paused longer at an AUG initiation codon present in strong context than one present in weak context. These results suggest that the dynamics of ribosome movement during translation of the reovirus s1 and s4 mRNAs are similar in vivo and in vitro and that the relative ribosome pause at an AUG initiation codon may be affected by the flanking nucleotide context of the AUG codon.
Doohan
J P
JP
Department of Biological Sciences, University of California, Santa Barbara 93106-9610.
Samuel
C E
CE
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002472:Cell Transformation, Viral; D003062:Codon / Q000235:genetics; D005814:Genes, Viral; D005822:Genetic Vectors; D007739:L Cells; D008715:Methionine / Q000378:metabolism; D051379:Mice; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis*; D012087:Reoviridae / Q000235:genetics* / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014158:Transcription, Genetic; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis*; D015678:Viral Structural Proteins / Q000235:genetics*
D003062:Codon; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; D014764:Viral Proteins; D015678:Viral Structural Proteins; D008715:Methionine