Projektdetails
Abstract
During environmental stress, bacteria alter their transcriptome and translatome to drastically reduce protein production to compensate for the energy-limitation. One possible mechanism to reduce translational activity is the activation of the endoribonuclease MazF that cleaves single stranded ACA sequences thereby resulting in bulk mRNA degradation. However, in a subset of mRNAs, MazF effectively removes the 5’-untranslated region without complete degradation, meanwhile also cleaving the 16S rRNA to remove the anti-Shine-Dalgarno (aSD) sequence. Hence, a distinct population of ribosomes, termed 70Sdelta43, is generated that translates the MazFprocessed mRNA, thereby globally regulating protein synthesis.
Due to the lack of the aSD and the presence of a 5’-terminal hydroxyl group on the mRNA that results from MazF cleavage, known to be inefficiently bound by wild-type 70S ribosomes, one would expect the interaction between 70Sdelta43 ribosomes and MazF-processed mRNA to be inefficient. However, 70Sdelta43 ribosomes still translate the truncated mRNAs suggesting cleavage removes a discriminatory element that generally differentiates against the 5’-hydroxyl end. This notion is supported by preliminary results, which indicate that 70Sdelta43 ribosomes do not distinguish between 5’-triphosphate and 5’-hydroxyl termini and form ternary complexes with either mRNA variant. In addition, we anticipate that auxiliary factors increase translation efficiency by stimulating this interaction. One likely candidate is elongation factor P, encoded by the efp gene, as it is part of the MazF regulon and has been shown to stimulate the formation of the first peptide bond. EF-P structurally mimics tRNA to influence the peptidyltransferase reaction and could therefore assist in the translation of mRNAs produced by MazF cleavage. I hypothesize that the 16S rRNA processing causes the 70Sdelta43 ribosome to promiscuously bind to truncated mRNAs regardless of the 5’-terminus phosphorylation state with this interaction further stabilized by EF-P. Furthermore, EF-P has been shown to bind to leaderless mRNAs that are part of the MazF regulon, and impact the translational efficiency of naturally leaderless mRNA cI, supporting its role in truncated mRNA initiation.
Thus, in the framework of this project, I will address the question of how mRNAs with various 5’-terminal groups are recognized by the 70Sdelta43 ribosome and whether additional factors, such as EF-P, assist this interaction. I plan to employ primer extension inhibition assays, ternary complex stability assays,
metabolic pulse labeling and RNAseq to address these questions.
This study aims to identify specific mRNA features that the 70Sdelta43 ribosomes can recognize to provide insight into controlling protein production during stress. The identity of the 5’-terminus appears to impact initiation
suggesting that 70S ribosomes scan mRNA termini which parallels the eukaryotic mode of translation initiation. Therefore, the results will provide novel information about ribosome evolution, intrinsic properties of the
translational machinery and post-transcriptional regulation due to ribosome heterogeneity during stress response.
Due to the lack of the aSD and the presence of a 5’-terminal hydroxyl group on the mRNA that results from MazF cleavage, known to be inefficiently bound by wild-type 70S ribosomes, one would expect the interaction between 70Sdelta43 ribosomes and MazF-processed mRNA to be inefficient. However, 70Sdelta43 ribosomes still translate the truncated mRNAs suggesting cleavage removes a discriminatory element that generally differentiates against the 5’-hydroxyl end. This notion is supported by preliminary results, which indicate that 70Sdelta43 ribosomes do not distinguish between 5’-triphosphate and 5’-hydroxyl termini and form ternary complexes with either mRNA variant. In addition, we anticipate that auxiliary factors increase translation efficiency by stimulating this interaction. One likely candidate is elongation factor P, encoded by the efp gene, as it is part of the MazF regulon and has been shown to stimulate the formation of the first peptide bond. EF-P structurally mimics tRNA to influence the peptidyltransferase reaction and could therefore assist in the translation of mRNAs produced by MazF cleavage. I hypothesize that the 16S rRNA processing causes the 70Sdelta43 ribosome to promiscuously bind to truncated mRNAs regardless of the 5’-terminus phosphorylation state with this interaction further stabilized by EF-P. Furthermore, EF-P has been shown to bind to leaderless mRNAs that are part of the MazF regulon, and impact the translational efficiency of naturally leaderless mRNA cI, supporting its role in truncated mRNA initiation.
Thus, in the framework of this project, I will address the question of how mRNAs with various 5’-terminal groups are recognized by the 70Sdelta43 ribosome and whether additional factors, such as EF-P, assist this interaction. I plan to employ primer extension inhibition assays, ternary complex stability assays,
metabolic pulse labeling and RNAseq to address these questions.
This study aims to identify specific mRNA features that the 70Sdelta43 ribosomes can recognize to provide insight into controlling protein production during stress. The identity of the 5’-terminus appears to impact initiation
suggesting that 70S ribosomes scan mRNA termini which parallels the eukaryotic mode of translation initiation. Therefore, the results will provide novel information about ribosome evolution, intrinsic properties of the
translational machinery and post-transcriptional regulation due to ribosome heterogeneity during stress response.
Status | Abgeschlossen |
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Tatsächlicher Beginn/ -es Ende | 1/02/18 → 31/01/20 |