Molecular Basis for Protein Synthesis by the Ribosome: Protein synthesis is the universal mechanism for translating the genetic code into cellular function. The machine that carries out translation is the ribosome, a large RNA-protein complex whose structure is highly conserved in all kingdoms of life. Ribosomes, which are over 20 nm in diameter, interact with several different ligands and cofactors, including messenger RNA (mRNA), transfer RNA (tRNA), and proteins involved in the initiation, elongation, and termination of protein synthesis. Ribosomes are also dynamic entities; the small and large ribosomal subunits associate and dissociate during one full cycle of protein synthesis. We are exploring the process of protein synthesis by the ribosome. Key questions about the fundamental nature of translation remain unanswered. For example, >b>how does the ribosome read the genetic code? And how do certain antibiotics, so useful in reducing infections, cripple ribosomes?
We are using x-ray crystallographic, biochemical, and genetic approaches to unravel the mechanism of protein synthesis. To investigate translational fidelity, we are studying 70S ribosomal complexes trapped in the process of choosing the correct aminoacyl tRNA. We are also probing the mechanism by which the ribosome translocates tRNAs from one binding site to the next after peptide bond formation. Many antibiotics degrade the accuracy of translation or prevent tRNA shuttling on the ribosome. We are looking at the structure of the bacterial ribosome both in the absence and presence of these antibiotics to decipher their effects on protein synthesis.
Two different bacteria are being used as the sources of ribosomes. First, we are using a thermophilic bacterium, since "extremophiles" have proven to be good sources for ribosomes in structural studies of translation. In collaboration with Marat Yusupov, Gulnara Yusupova, and Harry Noller, we are studying the structure of a 70S-ribosome complex with two tRNAs and mRNA at a nominal resolution of 5.5 Å (Fig. 1). We have modeled the ribosome based on the recently available atomic-resolution structures of the 30S and 50S subunits [N. Ban et al. (2000) Science 289, 905; B. Wimberly et al. (2000) Nature 407, 327]. The state of tRNA binding in this structure is probably that immediately prior to or immediately after peptidyl transfer. In collaboration with Professor Al Dahlberg and Dr. Steven Gregory at Brown University, we have made mutations in the ribosome which confer resistance or dependence on the antibiotic streptomycin. Streptomycin binds to ribosomes and leads to error-prone protein synthesis. The streptomycin resistance and dependence mutations in the ribosome do not disrupt streptomycin binding, yet are able to counteract the error-inducing effects of the antibiotic. We are looking at the structure of the intact mutant ribosomes, both in the absence and presence of the antibiotic, to decipher its effects on translation.
More recently, we have begun to study the structure of the ribosome from the common bacterium Escherichia coli. Many decades have been spent to develop both the biochemical and genetic tools to study protein synthesis in E. coli. We suspect, therefore, that ribosomes from this organism may provide the best means for determining the atomic-resolution structure of the ribosome in all stages of protein synthesis. In our preliminary work, we have obtained at least three crystal forms of 70S ribosomes from E. coli, and are in the process of determining the structure of the ribosome in each crystal form. Our crystallographic work with E. coli ribosomes is presently focused on the mechanism of tRNA selection, a process that involves mRNA, aminoacyl tRNA, elongation factor Tu, and the hydrolysis of GTP.
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