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)i 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.