Ribosome concentration contributes to discrimination against poly(A)- mRNA during translation initiation in Saccharomyces cerevisiae.
Inactivation of Saccharomyces cerevisiae poly(A) polymerase in a strain bearing the temperature-sensitive lethal pap1-1 mutation results in the synthesis of poly(A)- mRNAs that initiate translation with surprising efficiency. Translation of poly(A)- mRNAs after polyadenylation shut-off might result from an increase in the ratio of ribosomes and associated translation factors to mRNA, caused by the inability of poly(A)- mRNAs to accumulate to normal levels. To test this hypothesis, we used ribosomal subunit protein gene mutations to decrease either 40 or 60 S ribosomal subunit concentrations in strains carrying the pap1-1 mutation. Polyadenylation shut-off in such cells results in a nearly normal ratio of ribosomes to mRNA as revealed by polyribosome sedimentation analysis. Ribonuclease protection and Northern blot analyses showed that a significant percentage of poly(A)-deficient and poly(A)- mRNA associate with smaller polyribosomes compared with cells with normal ribosome levels. Analysis of the ratio of poly(A)-deficient and poly(A)- forms of a specific mRNA showed relatively more poly(A)- mRNA sedimenting with 20-60 S complexes than do poly(A)+ forms, suggesting a block in an early step of the translation initiation of the poly(A)- transcripts. These findings support models featuring the poly(A) tail as an enhancer of translation and suggest that the full effect of a poly(A) tail on the initiation strength of a mRNA may require competition for a limited number of free ribosomes or translation factors.
Proweller
A
A
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Butler
J S
JS
D004591:Electrophoresis, Polyacrylamide Gel; D000073718:Pancreatitis-Associated Proteins; D010442:Peptide Chain Initiation, Translational; D011061:Poly A; D011062:Polynucleotide Adenylyltransferase / Q000037:antagonists & inhibitors; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry*; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D012333:RNA, Messenger; D012269:Ribosomal Proteins; D011061:Poly A; D011062:Polynucleotide Adenylyltransferase