Regulation of translation rate is of crucial importance for such downstream co-translational pro- cesses as protein folding and modification, ligand binding, oligomerization, and interactions with chaperones and membranes. Although specific elements that modulate translation rate are well known, at present there is virtually a complete lack of results describing how such elements quantitatively affect translation rate. Our overall goal is to address this lack by quantifying the modulation of the rate of translation by E. coli ribosomes of specific mRNA and nascent peptide sequences, and to elucidate the mechanisms of such modulation. We use an approach coupling Total Internal Reflection Fluorescence Microscopy (TIRFM) with single molecule Fluorescence Resonance Energy Transfer (smFRET). Fluorescent reporter and smFRET pairs are introduced specifically into both aminoacyl tRNAs and ribosomal proteins L1 and L11, which are proximal to the tRNA E-site and A-site, respectively. The availability of these fluorescent-labeled compo- nents of the protein synthesis machinery permits observation on single ribosomes and in real time of FRET interactions monitoring aminoacyl-tRNA binding to the ribosomal A-site, interme- diate motions of tRNAs in adjacent sites, translocation of the tRNAs to the P- and E-sites, and release of discharged tRNA from the E-site. Single molecule assays have been developed both for short model mRNAs that emphasize pairing with fluorescent tRNAs and for mRNAs coding full-length proteins with the natural complement of all amino acids. Full expression of a rapidly maturing variant of green fluorescent protein, Emerald GFP (EmGFP), is signaled by fluores- cence of single EmGFP molecules in the TIRF microscope. Together, these approaches allow both discrimination of individual steps within the elongation cycle and continuous kinetic profiles of mRNA translation, providing unique insights into the regulation of translation rates in prokary- otic protein synthesis not accessible from other techniques. Our results will allow us to quantify the effects of modulatory elements and to determine if new elements and synergistic or antago- nistic effects are operative. Crucial for the correct interpretation of our results is the performance of control experiments demonstrating that the rate effects we measure result from introduction of the intended modulating element, rather than from involvement of one or more other ele- ments. We have 3 Specific Aims that focus on determining how the rhythm of protein synthesis is modulated by: 1. Codon usage and codon pair usage;2. Pause-inducing nascent peptides bound within the peptidyl transferase center of the ribosomes and the peptide exit tunnel;and 3. Pausing elements present in combination. Overall, our program of studying several pause ele- ments at the full protein expression level and within individual elongation cycles will define the magnitudes and detailed mechanisms of translation regulation.
Ribosome catalyzed protein synthesis is a core function of all living cells, but how it proceeds differs in detail between bacterial cells and eukaryotic, including mammalian, cells. These differ- ences are exploited in frequently utilized antibiotics, such as neomycin and erythromycin, which specifically target the bacterial ribosome and combat bacterial infections. However, widespread resistance to ribosomal antibiotics has limited their use. We are proposing sophisticated bio- chemical and biophysical studies to determine the precise functioning of bacterial ribosomes and how the rate and amount of expression is controlled by native and pathogen genomes. The knowledge gained from our experiments should both advance our basic understanding of how cells function and facilitate the development of new antibiotics for human use. Mechanisms re- vealed in the bacterial ribosome may either apply directly to eukaryotic protein synthesis or else help to inform further studies of the protein synthesis machinery of higher organisms. Specific mechanisms that are required for replication of viruses, such as programmed frameshifting, may someday be targets of therapy against cancer or AIDS.
|Rosenblum, Gabriel; Cooperman, Barry S (2014) Engine out of the chassis: cell-free protein synthesis and its uses. FEBS Lett 588:261-8|
|Liu, Wei; Kavaliauskas, Darius; Schrader, Jared M et al. (2014) Labeled EF-Tus for rapid kinetic studies of pretranslocation complex formation. ACS Chem Biol 9:2421-31|
|Chen, Chunlai; Zhang, Haibo; Broitman, Steven L et al. (2013) Dynamics of translation by single ribosomes through mRNA secondary structures. Nat Struct Mol Biol 20:582-8|
|Rosenblum, Gabriel; Chen, Chunlai; Kaur, Jaskiran et al. (2013) Quantifying elongation rhythm during full-length protein synthesis. J Am Chem Soc 135:11322-9|
|Liu, Wei; Shin, Dongwon; Tor, Yitzhak et al. (2013) Monitoring translation with modified mRNAs strategically labeled with isomorphic fluorescent guanosine mimetics. ACS Chem Biol 8:2017-23|
|Rosenblum, Gabriel; Chen, Chunlai; Kaur, Jaskiran et al. (2012) Real-time assay for testing components of protein synthesis. Nucleic Acids Res 40:e88|
|Chen, Chunlai; Stevens, Benjamin; Kaur, Jaskarin et al. (2011) Single-molecule fluorescence measurements of ribosomal translocation dynamics. Mol Cell 42:367-77|
|Bharill, Shashank; Chen, Chunlai; Stevens, Benjamin et al. (2011) Enhancement of single-molecule fluorescence signals by colloidal silver nanoparticles in studies of protein translation. ACS Nano 5:399-407|
|Chen, Chunlai; Stevens, Benjamin; Kaur, Jaskiran et al. (2011) Allosteric vs. spontaneous exit-site (E-site) tRNA dissociation early in protein synthesis. Proc Natl Acad Sci U S A 108:16980-5|
|Liu, Hanqing; Chen, Chunlai; Zhang, Haibo et al. (2011) The conserved protein EF4 (LepA) modulates the elongation cycle of protein synthesis. Proc Natl Acad Sci U S A 108:16223-8|
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