Functional Analysis of the Ribosome CAR Surface

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PROJECT SUMMARY/ABSTRACT Gene expression is controlled at multiple levels that are integrated so that cells, tissues and organisms can respond efficiently and quickly to changing environments. Regulated transcription of mRNAs is followed by integrated mechanisms of translational control to determine levels of protein production. Our proposed research is aimed at investigating a layer of translational regulation that depends upon a newly-identified ribosome interaction surface that is anchored to the ribosome A-site decoding center, allowing it to H-bond with the +1 codon that is next to enter the A site. This “CAR” interaction surface consists of three conserved residues—16S/18S rRNA C1054, A1196 (E. coli numbering) and ribosomal protein S3 R146 (S. cerevisiae numbering; conserved in eukaryotes)—and is anchored at the beginning of translocation to tRNA nucleotide (nt) 34, the anticodon partner of the codon wobble nucleotide. The CAR surface has the interesting property that its interactions are strongest when the +1 codon conforms to a GCN sequence. Since the initial codons— the ramp region—of a gene’s ORF are particularly influential in determining the rate at which ribosomes are launched down the mRNA, the extent to which ramp regions conform to GCN codons likely determines the sensitivity of each gene to this layer of regulation. An emerging idea in ribosome biology is that different versions of ribosomes are made under various cellular conditions, allowing cells to regulate protein translation according to their environments. Our analysis suggests that whether newly made ribosomes have modifications of various residues in the decoding center neighborhood—R146, one of the CAR residues, as well as several rRNA nucleotides—may determine whether the CAR interface can interact with the +1 codon. The enzymes for these modifications are down-regulated under stress conditions, and our molecular dynamics analysis indicates that when these modifications are present, the interaction of CAR with the +1 codon is considerably weaker. We will test if modification levels are reduced under stress in Aim 1. We also plan to examine ribosome behavior on mRNAs under stressed and unstressed conditions using ribosome profiling experiments to assess whether interactions between CAR and the +1 GCN codons may influence the rates at which ribosomes travel along the mRNA during translation (Aim 2). Since tRNA nt 34, which anchors CAR to the tRNA anticodon, often contains various modifications that are stress-dependent, we will carry out molecular dynamics analysis to test whether these modifications change anchoring of CAR to the anticodon with consequent changes in interactions of CAR with the +1 codon (Aim 3). This project, with its focus on integration of in vivo testing and computational modeling of ribosome dynamics, if successful, will lead to important insights into a new layer of translational regulation, and provide students with in-depth introductions to analysis of a system at the molecular level, as well as interdisciplinary modes of inquiry that reveal underlying behaviors of this biologically important system.