A Ribosome Interaction Surface Sensitive to mRNA GCN Periodicity

Scopino, Kristen; Williams, Elliot; Elsayed, Abdelrahman; Barr, William; Krizanc, Daniel; Thayer, Kelly M.; Weir, Michael P.

doi:10.1101/2020.04.28.058271

Cited by 5 publications

(28 citation statements)

References 43 publications

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“…Annotated ORFs in the yeast genome, aligned at their AUG start or stop codons (Fig 2A), show a 3-nucleotide GCN periodicity in which G at position 1 (G1) and C at position 2 (C2) of [27] of a subsystem of the translocating ribosome encompassing the A-site decoding center neighborhood showed that the +1 mRNA codon about to enter the A site H-bonds (red dashes) to the CAR interaction surface consisting of 16S/18S C1054, A1196 (E. coli numbering) and yeast ribosome protein Rps3 R146. Stacking interactions provide structural integrity to the CAR surface and anchor it to the wobble position anticodon nucleotide (tRNA nucleotide 34).…”

Section: Gcn Periodicity Is Pronounced In the 5' Ramp Region Of Highlmentioning

confidence: 99%

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GCN sensitive protein translation in yeast

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Levels of protein translation by ribosomes are governed both by features of the translation machinery as well as sequence properties of the mRNAs themselves. We focus here on a striking three-nucleotide periodicity, characterized by overrepresentation of GCN codons and underrepresentation of G at the second position of codons, that is observed in Open Reading Frames (ORFs) of mRNAs. Our examination of mRNA sequences in Saccharomyces cerevisiae revealed that this periodicity is particularly pronounced in the initial codonsthe ramp region-of ORFs of genes with high protein expression. It is also found in mRNA sequences immediately following non-standard AUG start sites, located upstream or downstream of the standard annotated start sites of genes. To explore the possible influences of the ramp GCN periodicity on translation efficiency, we tested edited ramps with accentuated or depressed periodicity in two test genes, SKN7 and HMT1. Greater conformance to (GCN) n was found to significantly depress translation, whereas disrupting conformance had neutral or positive effects on translation. Our recent Molecular Dynamics analysis of a subsystem of translocating ribosomes in yeast revealed an interaction surface that H-bonds to the +1 codon that is about to enter the ribosome decoding center A site. The surface, comprised of 16S/18S rRNA C1054 and A1196 (E. coli numbering) and R146 of ribosomal protein Rps3, preferentially interacts with GCN codons, and we hypothesize that modulation of this mRNA-ribosome interaction may underlie GCN-mediated regulation of protein translation. Integration of our expression studies with large-scale reporter studies of ramp sequence variants suggests a model in which the C1054-A1196-R146 (CAR) interaction surface can act as both an accelerator and braking system for ribosome translation.

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Section: Gcn Periodicity Is Pronounced In the 5' Ramp Region Of Highlmentioning

confidence: 99%

“… Molecular Dynamics analysis [ 27 ] of a subsystem of the translocating ribosome encompassing the A-site decoding center neighborhood showed that the +1 mRNA codon about to enter the A site H-bonds (red dashes) to the CAR interaction surface consisting of 16S/18S C1054, A1196 ( E . coli numbering) and yeast ribosome protein Rps3 R146.…”

Section: Introductionmentioning

confidence: 99%

GCN sensitive protein translation in yeast

et al. 2020

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“…As introduced earlier, we have identified [27] a ribosome interaction surface located next to the decoding center A site that may mediate sensitivity to the ramp GCN periodicity (Fig 1). At the beginning of translocation, the C1054-A1196-R146 (CAR) interaction surface is anchored through base stacking to the wobble anticodon nucleotide (nucleotide 34) of the A-site tRNA, aligning the CAR surface so that it can H-bond with the +1 codon about to enter the A site.…”

Section: The Car Surface May Mediate Gcn Sensitivitymentioning

confidence: 80%

“…However, crystal and cryo-EM ribosome structures have not shown base pairing between the 530 loop rRNA and mRNA. Molecular Dynamics analysis [27] of a subsystem of the translocating ribosome encompassing the Asite decoding center neighborhood showed that the +1 mRNA codon about to enter the A site H-bonds (red dashes) to the CAR interaction surface consisting of 16S/18S C1054, A1196 (E. coli numbering) and yeast ribosome protein Rps3 R146. Stacking interactions provide structural integrity to the CAR surface and anchor it to the wobble position anticodon nucleotide (tRNA nucleotide 34).…”

Section: Introductionmentioning

confidence: 99%

“…The structure is viewed with Visual Molecular Dynamics (VMD; [28,29]). Given these intriguing observations, we decided to examine the dynamic behavior of a subsystem of the ribosome centered around the A-site decoding center and 530 loop neighborhood using Molecular Dynamics (MD) simulations [27]. These MD experiments have shown that rRNA nucleotides in the 530 loop tend to stay base paired with each other in a standard configuration.…”

Section: Introductionmentioning

confidence: 99%

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GCN sensitive protein translation in yeast

Barr¹,

Sheth²,

Kwon³

et al. 2020

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MPW) ¶ These authors contributed equally to this work. AbstractLevels of protein translation by ribosomes are governed both by features of the translation machinery as well as sequence properties of the mRNAs themselves. We focus here on a striking three-nucleotide periodicity, characterized by overrepresentation of GCN codons and underrepresentation of G at the second position of codons, that is observed in Open Reading Frames (ORFs) of mRNAs. Our examination of mRNA sequences in Saccharomyces cerevisiae revealed that this periodicity is particularly pronounced in the initial codons--the ramp region--of ORFs of genes with high protein expression. It is also found in mRNA sequences immediately following non-standard AUG start sites, located upstream or downstream of the standard annotated start sites of genes. To explore the possible influences of the ramp GCN periodicity on translation efficiency, we tested edited ramps with accentuated or depressed periodicity in two test genes, SKN7 and HMT1. Greater conformance to (GCN) n was found to significantly depress translation, whereas disrupting conformance had neutral or positive effects on translation. Our recent Molecular Dynamics analysis of a subsystem of translocating ribosomes in yeast revealed an interaction surface that H-bonds to the +1 codon that is about to enter the ribosome decoding center A site. The surface, comprised of 16S/18S rRNA C1054 and A1196 (E. coli numbering) and R146 of ribosomal protein Rps3, preferentially interacts with GCN codons, and we hypothesize that modulation of this mRNA-ribosome interaction may underlie GCN-mediated regulation of protein translation. Integration of our expression studies with largescale reporter studies of ramp sequence variants suggests a model in which the C1054-A1196-R146 (CAR) interaction surface can act as both an accelerator and braking system for ribosome translation.

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The CAR mRNA-Interaction Surface is a Zipper Extension of the Ribosome A Site

Dalgarno

Scopino

Raval

et al. 2022

Preprint

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The ribosome CAR interaction surface behaves like an extension of the decoding center A site and has H-bond interactions with the +1 codon that is next in line to enter the A site. Through molecular dynamics simulations, we investigated the codon sequence specificity of this CAR-mRNA interaction and discovered a strong preference for GCN codons, suggesting that there may be a sequence-dependent layer of translational regulation dependent on the CAR interaction surface. Dissection of the CAR-mRNA interaction through nucleotide substitution experiments showed that the first nucleotide of the +1 codon dominates over the second nucleotide position, consistent with an energetically favorable zipper-like activity that emanates from the A site through the CAR-mRNA interface. The +1 codon/CAR interaction is also affected by the identity of nucleotide 3 of +1 GCN codons which influences the stacking of G and C. Clustering analysis suggests that the A site decoding center adopts different neighborhood substates that depend on the identity of the +1 codon.

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A Ribosome Interaction Surface Sensitive to mRNA GCN Periodicity

Cited by 5 publications

References 43 publications

GCN sensitive protein translation in yeast

GCN sensitive protein translation in yeast

GCN sensitive protein translation in yeast

The CAR mRNA-Interaction Surface is a Zipper Extension of the Ribosome A Site

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