Domain III of Saccharomyces cerevisiae 25 S ribosomal RNA: its role in binding of ribosomal protein L25 and 60 S subunit formation.
Domain III of Saccharomyces cerevisiae 25 S rRNA contains the recognition site for the primary rRNA-binding ribosomal protein L25, which belongs to the functionally conserved EL23/L25 family of ribosomal proteins. The EL23/L25 binding region is very complex, consisting of several irregular helices held together by long-distance secondary and tertiary interactions. Moreover, it contains the eukaryote-specific V9 (D7a) expansion segment. Functional characterisation of the structural elements of this site by a detailed in vitro and in vivo mutational analysis indicates the presence of two separate regions that are directly involved in L25 binding. In particular, mutation of either of two conserved nucleotides in the loop of helix 49 significantly reduces in vitro L25 binding, thus strongly supporting their role as attachment sites for the r-protein. Two other helices appear to be primarily required for the correct folding of the binding site. Mutations that abolish in vitro binding of L25 block accumulation of 25 S rRNA in vivo because they stall pre-rRNA processing at the level of its immediate precursor, the 27 S(B) pre-rRNA. Surprisingly, several mutations that do not significantly affect L25 binding in vitro cause the same lethal defect in 27 S(B) pre-rRNA processing. Deletion of the V9 expansion segment also leads to under-accumulation of mature 25 S rRNA and a twofold reduction in growth rate. We conclude that an intact domain III, including the V9 expansion segment, is essential for normal processing and assembly of 25 S rRNA.
van Beekvelt
C A
CA
Department of Biochemistry and Molecular Biology, IMBW BioCentrum Amsterdam, Vrije Universiteit, de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Kooi
E A
EA
de Graaff-Vincent
M
M
Riet
J
J
Venema
J
J
Raué
H A
HA
D001483:Base Sequence; D001665:Binding Sites; D002455:Cell Division; D017124:Conserved Sequence / Q000235:genetics; D005804:Genes, Lethal / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012335:RNA, Ribosomal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism
D012322:RNA Precursors; D012331:RNA, Fungal; D012335:RNA, Ribosomal; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C031127:ribosomal protein L25; C065039:RNA, ribosomal, 25S