Extending the Spacing between the Shine–Dalgarno Sequence and P-Site Codon Reduces the Rate of mRNA Translocation

Wakabayashi, Hironao; Warnasooriya, Chandani; Ermolenko, Dmitri N.

doi:10.1016/j.jmb.2020.06.008

Cited by 3 publications

(2 citation statements)

References 41 publications

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“…The only clear asymmetry at the level of RNA is the Shine–Dalgarno sequence. This stretch of nucleotides was hypothesized to hamper early elongation events because it binds to the ribosome 58 , an effect that has been recently verified experimentally 59 . Also, DS sequestration by early codons through stem-loop formation may occur 28 .…”

Section: Discussionmentioning

confidence: 89%

Peptidyl transferase center decompaction and structural constraints during early protein elongation on the ribosome

Jia

Wang

Lehmann

2021

Sci Rep

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Peptide bond formation on the ribosome requires that aminoacyl-tRNAs and peptidyl-tRNAs are properly positioned on the A site and the P site of the peptidyl transferase center (PTC) so that nucleophilic attack can occur. Here we analyse some constraints associated with the induced-fit mechanism of the PTC, that promotes this positioning through a compaction around the aminoacyl ester orchestrated by U2506. The physical basis of PTC decompaction, that allows the elongated peptidyl-tRNA to free itself from that state and move to the P site of the PTC, is still unclear. From thermodynamics considerations and an analysis of published ribosome structures, the present work highlights the rational of this mechanism, in which the free-energy released by the new peptide bond is used to kick U2506 away from the reaction center. Furthermore, we show the evidence that decompaction is impaired when the nascent peptide is not yet anchored inside the exit tunnel, which may contribute to explain why the first rounds of elongation are inefficient, an issue that has attracted much interest for about two decades. Results in this field are examined in the light of the present analysis and a physico-chemical correlation in the genetic code, which suggest that elementary constraints associated with the size of the side-chain of the amino acids penalize early elongation events.

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Section: Discussionmentioning

confidence: 89%

Peptidyl transferase center decompaction and structural constraints during early protein elongation on the ribosome

Jia

Wang

Lehmann

2021

Sci Rep

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“…High GC content in the 5' UTR might form a more stable mRNA secondary structure, which can reduce the translation initiation ef iciency as well [25]. Furthermore, since the long spacer sequence resulted in a mismatch of start codon and ribosome, the combination of fMet-tRNA was weakened as well [26]. Therefore, in this work, the addition of the extra CG sequence in the spacer sequence resulted in a low rate of translation initiation, and further reduced expression level.…”

Section: The Eff Ect Of Expression Level On the Functional Expression...mentioning

confidence: 99%

Enhancing functional expression of L-glycerophosphate oxidase in Escherichia coli by controlling the expression rate

Zhang¹,

Huanbo²,

Du³

et al. 2022

Ann Biomed Sci Eng

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Heterologous expression of proteins often pursues high expression levels, but it can easily result in misfolding and loss of biological function. L-α-glycerophosphate oxidase (GlpO) is a flavin adenine dinucleotide (FAD)-dependent oxidase which is widely used in the clinical determination of triglycerides. We found that the total enzymatic activity of GlpO expressed in Escherichia coli (E. coli) was extremely low, probably due to the absence of FAD cofactors and the misfolding of GlpO at a high synthesis rate. Therefore, decreasing the expression rate was used to improve the activity of GlpO. The specific activity of GlpO expressed on the pUC19 vector with lac promotor was approximately 30 times higher than that expressed on the pET28a vector with T7 promotor, but the expression levels of GlpO on the two vectors were completely opposite. It indicated that the specific activity of GlpO was increased as the expression level decreased. However, too low expression greatly influences the total amount and activity of the functional enzyme. In order to resolve this problem, two new plasmids, GlpO-CG4 and GlpO-CG6, were constructed by inserting 4 or 6 nucleotides, respectively, between the ribosome binding site (RBS) and the start code (ATG) on pET28a. Compared with the expression on the GlpO-pET vector, the expression rates of GlpO on the GlpO-CG4 and GlpO-CG6 were dramatically decreased. The total activity of GlpO expressed on GlpO-CG6 was 11 times and 1.5 times higher than that expressed on the GlpO-pET and GlpO-pUC, respectively. Results suggest that the activity of GlpO can be improved by decreasing the expression rate.

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Ribosome heterogeneity results in leader sequence-mediated regulation of protein synthesis in Francisella tularensis

Trautmann,

Schmidt,

Gregory

et al. 2023

J Bacteriol

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Although ribosomes are generally examined in aggregate, ribosomes can be heterogenous in composition. Evidence is accumulating that changes in ribosome composition may result in altered function, such that ribosome heterogeneity may provide a mechanism to regulate protein synthesis. Ribosome heterogeneity in the human pathogen Francisella tularensis results from incorporation of one of three homologs of bS21, a small ribosomal subunit protein demonstrated to regulate protein synthesis in other bacteria. Loss of one homolog, bS21-2, results in genome-wide post-transcriptional changes in protein abundance. This suggests that bS21-2 can, either directly or indirectly, lead to preferential translation of particular mRNAs. Here, we examine the potential of bS21-2 to function in a leader sequence-dependent manner and to function indirectly, via Hfq. We found that the 5´ untranslated region (UTR) of some bS21-2-responsive genes, including key virulence genes, is sufficient to alter translation in cells lacking bS21-2. We further identify features of a 5´ UTR that allow responsiveness to bS21-2. These include an imperfect Shine-Dalgarno sequence and a particular six nucleotide sequence. Our results are consistent with a model in which a bS21 homolog increases the efficiency of translation initiation through interactions with specific leader sequences. With respect to bS21-2 indirectly regulating translation via the RNA-binding protein Hfq, we found that Hfq controls transcript abundance rather than protein synthesis, impacting virulence gene expression via a distinct mechanism. Together, we determined that ribosome composition in F. tularensis regulates translation in a leader sequence-dependent manner, a regulatory mechanism which may be used in other bacteria. IMPORTANCE Ribosome heterogeneity is common in bacteria, and there is mounting evidence that ribosome composition plays a regulatory role in protein synthesis. However, mechanisms of ribosome-driven gene regulation are not well understood. In the human pathogen Francisella tularensis , which encodes multiple homologs for the ribosomal protein bS21, loss of one homolog impacts protein synthesis and virulence. Here, we explore the mechanism behind bS21-mediated changes in protein synthesis, finding that they can be linked to altered translation initiation and are dependent on specific sequences in the leaders of transcripts. Our data support a model in which ribosome composition regulates gene expression through translation, a strategy that may be conserved in diverse organisms with various sources of ribosome heterogeneity.

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Extending the Spacing between the Shine–Dalgarno Sequence and P-Site Codon Reduces the Rate of mRNA Translocation

Cited by 3 publications

References 41 publications

Peptidyl transferase center decompaction and structural constraints during early protein elongation on the ribosome

Peptidyl transferase center decompaction and structural constraints during early protein elongation on the ribosome

Enhancing functional expression of L-glycerophosphate oxidase in Escherichia coli by controlling the expression rate

Ribosome heterogeneity results in leader sequence-mediated regulation of protein synthesis in Francisella tularensis

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