The final step of gene expression is translation, the synthesis of protein from the information content of messenger RNA (mRNA). Translation occurs in ribosomes, subcellular machines composed of both ribosomal RNA (rRNA) and protein. In recent years, x-ray crystallography and cryo-electron microscopy have revealed the structure of the ribosome and its interactions with a variety of substrates and translation factors. Despite this wealth of structural information, questions of how the ribosome facilitates specific events during translation remain unclear. The goal of this project is to better understand the initiation phase of translation. Initiation is a complex process that requires selection of the initiator tRNA from other species of tRNA and selection of the start codon from other similar or identical triplet sequences in the mRNA. In bacteria, the process is kinetically controlled by initiation factors IF1, IF2 and IF3. In other organisms, initiation is fundamentally similar but more elaborate, involving 10 or more factors in addition to universally conserved IF2 (eIF5B) and IF1 (eIF1A). The Fredrick lab has identified a number of mutations in the rRNA of the small subunit that cause defects in start codon selection. This project is centered on the analysis of these mutations, which will help elucidate the role of rRNA and factors IF1 and IF3 in the initiation process.
The broader impact of this project lies in its strong educational emphasis. This project will not only provide research training for both undergraduate and graduate students but will also give research opportunities for secondary educators. Each summer, the laboratory will host a high school science teacher from one of the Columbus Public Schools. While contributing to the research efforts of the laboratory, these teachers will gain experience with modern molecular biology to strengthen their curriculum.
In all organisms, the genetic code is read by ribosomes, large RNA-based enzymes. Ribosomes synthesize proteins based on the information content of messenger RNA (mRNA) templates, which are copied from the genomic DNA. Over the past decade, tremendous progress has been made in understanding the structure of the ribosome. Yet, how the various parts of the ribosome participate in the steps of protein synthesis remains unclear. This project was focused on initiation of translation, which entails the rapid and accurate assembly of a ribosomal complex containing the initiator tRNA (fMet-tRNA in bacteria) paired to the start codon of the mRNA in the peptidyl (P) site. The overall research goal of the project was to better understand how the small (30S) subunit of the ribosome participates in the process, in conjunction with the protein initiation factors IF1 and IF3, to ensure accurate start codon selection. It was found that a specific region of the subunit (A1408 region of helix h44 of 16S rRNA) changes conformation in response to start codon recognition, and this conformational change promotes docking of the large (50S) subunit (i.e., productive initiation). In the absence of the correct start codon, this change does not occur and the pathway is inhibited. It was also shown that the 30S P site can discriminate the correct start codon (AUG) from similar codons (such as AUC) by a factor of > 1000, in the absence of protein factors. This exceeds the level of discrimination seen for initiation in the cell by 10-fold, suggesting that the factors act to speed the reaction without increasing the inherent selectivity of the P site. These findings shed new light on translation initiation, an important aspect of gene expression. This project achieved broader impact by (1) providing summer internships to high school students of Columbus public schools, (2) integrating real science (novel genetic screens) into the curriculum of upper-level undergraduate laboratory courses at the Ohio State University, and (3) participating in the OSTEP Summer Bridge Program, which aims to help new freshman science majors transition successfully into their college curriculum.
