Ribosome Concentration Contributes to Discrimination against Poly(A)− mRNA during Translation Initiation in Saccharomyces cerevisiae

• Published in: The Journal of biological chemistry Vol. 272; no. 9; pp. 6004 - 6010
• Main Authors: Proweller, Aaron, Butler, J.Scott
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 28.02.1997; American Society for Biochemistry and Molecular Biology
• Subjects: ARN MENSAJERO; ARN MESSAGER; CHEMICAL REACTIONS; Electrophoresis, Polyacrylamide Gel; GENE; GENES; MESSENGER RNA; MUTACION; MUTATION; PAB1 GENE; Pancreatitis-Associated Proteins; Peptide Chain Initiation, Translational; PGK1 GENE; Poly A; POLY(A) POLYMERASE; POLYADENYLATED MESSENGER RNA; POLYADENYLATION; Polynucleotide Adenylyltransferase - antagonists & inhibitors; POLYRIBOSOMES; Protein Biosynthesis; REACCIONES QUIMICAS; REACTION CHIMIQUE; Ribosomal Proteins - genetics; Ribosomal Proteins - metabolism; RIBOSOMAS; RIBOSOME; RIBOSOMES; Ribosomes - metabolism; RNA POLYMERASE; RNA, Messenger - chemistry; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - genetics; TCM1 GENE; TRANSFERASAS; TRANSFERASE; TRANSFERASES
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.9.6004

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.