Protein synthesis by the ribosome is central to life and disease. We have developed single- molecule approaches to provide a real-time dynamic perspective to the basic and unusual steps of protein synthesis. Here we build on our methodological developments over the past funding period to provide combined structural and dynamic view of how mRNA and nascent protein sequence and structure modulate translation and affect its outcome.
In Aim 1 we focus initiation and termination. We will understand the conformational dynamics of the 30S subunit during initiation and how the process is controlled by mRNA sequence and structure; we will study termination regulation by RF3, and recoding in selenocysteine incorporation; finally, we will watch how polysomes form.
In Aim 2, we will study the role of mRNA sequence and structure in controlling elongation. We will investigate how modified nucleotides in mRNA change elongation, and study how ribosomes navigate secondary structures. We will determine the basis for pausing and reading-frame changes that occur during elongation due to mRNA sequence or structure, tracking pathways, branchpoints and mechanism. We will also determine how two ribosomes translating on an RNA navigate secondary structures and possibly interact during elongation.
In Aim 3, we explore the interplay of nascent protein sequence and folding with elongation kinetics. We will determine how antibiotic sensitivity or resistance is determined by the nascent chain, and how translation is disrupted. We will determine how the folding of well-characterized protein domains on the ribosome affect real time translation (pausing, stalling acceleration). Finally, we will explore the directly the correlation of folding and translation in real time. The proposed research is buttressed by strong collaborations on the various systems to support biophysical and structural analysis, reagent preparation, and in vivo correlation. The results of this proposal will provide a deeply textured view for how protein synthesis is regulated and controlled.
Translation of proteins by the ribosome in central to life. Here we observe ribosomes translating in real time, animating the process of protein synthesis. We will obtain a combined structural and dynamic view how translation is controlled by RNA sequence and structure, and by the nature and folding of the protein products.
|Choi, Junhong; Indrisiunaite, Gabriele; DeMirci, Hasan et al. (2018) 2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation. Nat Struct Mol Biol 25:208-216|
|Prabhakar, Arjun; Capece, Mark C; Petrov, Alexey et al. (2017) Post-termination Ribosome Intermediate Acts as the Gateway to Ribosome Recycling. Cell Rep 20:161-172|
|Choi, Junhong; Puglisi, Joseph D (2017) Three tRNAs on the ribosome slow translation elongation. Proc Natl Acad Sci U S A 114:13691-13696|
|Prabhakar, Arjun; Choi, Junhong; Wang, Jinfan et al. (2017) Dynamic basis of fidelity and speed in translation: Coordinated multistep mechanisms of elongation and termination. Protein Sci 26:1352-1362|
|Navon, Sharon Penias; Kornberg, Guy; Chen, Jin et al. (2016) Amino acid sequence repertoire of the bacterial proteome and the occurrence of untranslatable sequences. Proc Natl Acad Sci U S A 113:7166-70|
|Sierra, Raymond G; Gati, Cornelius; Laksmono, Hartawan et al. (2016) Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II. Nat Methods 13:59-62|
|Choi, Junhong; Ieong, Ka-Weng; Demirci, Hasan et al. (2016) N(6)-methyladenosine in mRNA disrupts tRNA selection and translation-elongation dynamics. Nat Struct Mol Biol 23:110-5|
|Petrov, Alexey; Grosely, Rosslyn; Chen, Jin et al. (2016) Multiple Parallel Pathways of Translation Initiation on the CrPV IRES. Mol Cell 62:92-103|
|Puglisi, Joseph D (2015) Synthetic biology: Ribosomal ties that bind. Nature 524:45-6|
|Chen, Jin; Coakley, Arthur; O'Connor, Michelle et al. (2015) Coupling of mRNA Structure Rearrangement to Ribosome Movement during Bypassing of Non-coding Regions. Cell 163:1267-1280|
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