282970 A Biophysical Model of Structured Ribosomal Standby Sites and Their Effect On the mRNA Translation Rate

Wednesday, October 31, 2012: 9:24 AM
Westmoreland Central (Westin )
Amin Espah Borujeni, Chemical Engineering, Pennsylvania State University, State College, PA and Howard Salis, Chemical Engineering / Biological Engineering, Pennsylvania State University, University Park, PA

Translation initiation is a key rate-limiting step during protein expression. It begins when the ribosome's 30S subunit initially binds to the mRNA at what is now called the ribosomal standby site. After initial contact, the 30S subunit shifts into position over the ribosome binding site, followed by initiation of polypeptide synthesis.  We previously developed a thermodynamic model, called the Ribosome Binding Site Calculator, that predicts the Gibbs free energy change when the ribosome binds to the mRNA. Using statistical thermodynamics, this Gibbs free energy change is then related to the mRNA's translation initiation rate. The previous model made strong assumptions about the nature of the ribosomal standby site and how mRNA structures upstream of the ribosome binding site affect the rate of translation initiation. Accordingly, the model was less accurate when structured ribosomal standby sites were present. Structured standby sites are often present within bacterial operons, and can facilitate post-transcriptional regulation of protein expression.

We present an exhaustive quantitative characterization that establishes how a structured ribosomal standby site affects the rate of translation initiation. Using this experimental data, we develop a biophysical model that predicts the Gibbs free energy of ribosome binding to an arbitrarily structured ribosomal standby site. The thermodynamic model accounts for diverse mRNA structures with varying hairpin sizes and folding energies, and proximal and distal standby sites with varying lengths. The model's predictions were experimentally validated by employing flow cytometry to measure fluorescent protein expression from over 90 designed, synthetic mRNA sequences, in addition to mRNA level measurements with RT-qPCR.

We present two key findings:  (i)  the ribosome's 30S subunit does not unfold strong mRNA structures surrounding standby sites and will instead bind to constrained single-stranded regions via compression; and (ii) the ribosome can bind to distal standby sites and bypass mRNA structures via rolling or sliding to shift into position over a ribosome binding site. Our findings show that the ribosome is a flexible molecular machine that minimizes its binding free energy to mRNA.


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