Ribosomal protein L33 is required for ribosome biogenesis, subunit joining, and repression of GCN4 translation.
We identified a mutation in the 60S ribosomal protein L33A (rpl33a-G76R) that elicits derepression of GCN4 translation (Gcd- phenotype) by allowing scanning preinitiation complexes to bypass inhibitory upstream open reading frame 4 (uORF4) independently of prior uORF1 translation and reinitiation. At 37 degrees C, rpl33a-G76R confers defects in 60S biogenesis comparable to those produced by the deletion of RPL33A (DeltaA). At 28 degrees C, however, the 60S biogenesis defect is less severe in rpl33a-G76R than in DeltaA cells, yet rpl33a-G76R confers greater derepression of GCN4 and a larger reduction in general translation. Hence, it appears that rpl33a-G76R has a stronger effect on ribosomal-subunit joining than does a comparable reduction of wild-type 60S levels conferred by DeltaA. We suggest that rpl33a-G76R alters the 60S subunit in a way that impedes ribosomal-subunit joining and thereby allows 48S rRNA complexes to abort initiation at uORF4, resume scanning, and initiate downstream at GCN4. Because overexpressing tRNAiMet suppresses the Gcd- phenotype of rpl33a-G76R cells, dissociation of tRNAiMet from the 40S subunit may be responsible for abortive initiation at uORF4 in this mutant. We further demonstrate that rpl33a-G76R impairs the efficient processing of 35S and 27S pre-rRNAs and reduces the accumulation of all four mature rRNAs, indicating an important role for L33 in the biogenesis of both ribosomal subunits.
Martín-Marcos
Pilar
P
Instituto de Microbiología Bioquímica, CSIC/Universidad de Salamanca, Edificio Departamental de Biología, Campus Miguel de Unamuno, 37007 Salamanca, Spain.
Hinnebusch
Alan G
AG
Tamame
Mercedes
M
D000595:Amino Acid Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D010641:Phenotype; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C501391:GCN4 protein, S cerevisiae; C069566:MRPL33 protein, S cerevisiae; D021122:Protein Subunits; D012322:RNA Precursors; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors