Dataset Information

Ribosomal Protein S12 Hastens Nucleation of Co-Transcriptional Ribosome Assembly.

ABSTRACT: Ribosomal subunits begin assembly during transcription of the ribosomal RNA (rRNA), when the rRNA begins to fold and associate with ribosomal proteins (RPs). In bacteria, the first steps of ribosome assembly depend upon recognition of the properly folded rRNA by primary assembly proteins such as S4, which nucleates assembly of the 16S 5' domain. Recent evidence, however, suggests that initial recognition by S4 is delayed due to variable folding of the rRNA during transcription. Here, using single-molecule colocalization co-transcriptional assembly (smCoCoA), we show that the late-binding RP S12 specifically promotes the association of S4 with the pre-16S rRNA during transcription, thereby accelerating nucleation of 30S ribosome assembly. Order of addition experiments suggest that S12 helps chaperone the rRNA during transcription, particularly near the S4 binding site. S12 interacts transiently with the rRNA during transcription and, consequently, a high concentration is required for its chaperone activity. These results support a model in which late-binding RPs moonlight as RNA chaperones during transcription in order to facilitate rapid assembly.

SUBMITTER: Rodgers ML

PROVIDER: S-EPMC10296100 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Publications

Ribosomal Protein S12 Hastens Nucleation of Co-Transcriptional Ribosome Assembly.

Rodgers Margaret L ML Sun Yunsheng Y Woodson Sarah A SA

Biomolecules 20230606 6

Ribosomal subunits begin assembly during transcription of the ribosomal RNA (rRNA), when the rRNA begins to fold and associate with ribosomal proteins (RPs). In bacteria, the first steps of ribosome assembly depend upon recognition of the properly folded rRNA by primary assembly proteins such as S4, which nucleates assembly of the 16S 5' domain. Recent evidence, however, suggests that initial recognition by S4 is delayed due to variable folding of the rRNA during transcription. Here, using singl ...[more]

PMID: 37371531

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Project description:The eukaryotic ribosome consists of a small (40S) and a large (60S) subunit. Rps26 is one of the essential ribosomal proteins of the 40S subunit and is encoded by two almost identical genes, RPS26a and RPS26b. Previous studies demonstrated that Rps26 interacts with the 5' untranslated region of mRNA via the eukaryote-specific 62-YXXPKXYXK-70 (Y62-K70) motif. Those observations suggested that this peptide within Rps26 might play an important and specific role during translation initiation. By using alanine-scanning mutagenesis and engineered strains of the yeast Saccharomyces cerevisiae, we found that single amino acid substitutions within the Y62-K70 motif of Rps26 did not affect the in vivo function of the protein. In contrast, complete deletion of the Y62-K70 segment was lethal. The simultaneous replacement of five conserved residues within the Y62-K70 segment by alanines resulted in growth defects under stress conditions and produced distinct changes in polysome profiles that were indicative of the accumulation of free 60S subunits. Human Rps26 (Rps26-Hs), which displays significant homology with yeast Rps26, supported the growth of an S. cerevisiae Δrps26a Δrps26b strain. However, the Δrps26a Δrps26b double deletion strain expressing Rps26-Hs displayed substantial growth defects and an altered ratio of 40S/60S ribosomal subunits. The combined data strongly suggest that the eukaryote-specific motif within Rps26 does not play a specific role in translation initiation. Rather, the data indicate that Rps26 as a whole is necessary for proper assembly of the 40S subunit and the 80S ribosome in yeast. IMPORTANCE Rps26 is an essential protein of the eukaryotic small ribosomal subunit. Previous experiments demonstrated an interaction between the eukaryote-specific Y62-K70 segment of Rps26 and the 5' untranslated region of mRNA. The data suggested a specific role of the Y62-K70 motif during translation initiation. Here, we report that single-site substitutions within the Y62-K70 peptide did not affect the growth of engineered yeast strains, arguing against its having a critical role during translation initiation via specific interactions with the 5' untranslated region of mRNA molecules. Only the simultaneous replacement of five conserved residues within the Y62-K70 fragment or the replacement of the yeast protein with the human homolog resulted in growth defects and caused significant changes in polysome profiles. The results expand our knowledge of ribosomal protein function and suggest a role of Rps26 during ribosome assembly in yeast.

Dataset Information

Ribosomal Protein S12 Hastens Nucleation of Co-Transcriptional Ribosome Assembly.

Publications

Ribosomal Protein S12 Hastens Nucleation of Co-Transcriptional Ribosome Assembly.

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