ja405205c_si_003.mpg (2.36 MB)
Quantifying Elongation Rhythm during Full-Length Protein Synthesis
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posted on 2013-07-31, 00:00 authored by Gabriel Rosenblum, Chunlai Chen, Jaskiran Kaur, Xiaonan Cui, Haibo Zhang, Haruichi Asahara, Shaorong Chong, Zeev Smilansky, Yale E. Goldman, Barry S. CoopermanPauses regulate the rhythm of ribosomal
protein synthesis. Mutations
disrupting even minor pauses can give rise to improperly formed proteins
and human disease. Such minor pauses are difficult to characterize
by ensemble methods, but can be readily examined by single-molecule
(sm) approaches. Here we use smFRET to carry out real-time monitoring
of the expression of a full-length protein, the green fluorescent
protein variant Emerald GFP. We demonstrate significant correlations
between measured elongation rates and codon and isoacceptor tRNA usage,
and provide a quantitative estimate of the effect on elongation rate
of replacing a codon recognizing an abundant tRNA with a synonymous
codon cognate to a rarer tRNA. Our results suggest that tRNA selection
plays an important general role in modulating the rates and rhythms
of protein synthesis, potentially influencing simultaneous co-translational
processes such as folding and chemical modification.