RESEARCH @ PATRICIA L. CLARK LAB
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Co-translational conformations of nascent chains
We use a wide variety of techniques to study the conformations of newly-synthesized polypeptide chains while they are still undergoing synthesis by the ribosome. We pioneered a technique to transiently pause ribosome translation at a specific point by adapting a naturally-occurring pause sequence from an E. coli protein, SecM. We have used this technique to determine that co-translational conformations of nascent chains are distinct from the conformations populated during in vitro refolding of purified protein (either full length, or C-terminally truncated in order to more closely resemble the nascent chain). We have developed novel methods to detect nascent chain conformations, and adapted existing methodology. Read more about these studies:
Ugrinov K.G. & Clark P.L. (2010) Co-translational folding increases GFP folding yield. Biophysical Journal 98, 1312-1320. [PDF]
Clark P.L. & Ugrinov K.G (2009) Measuring co-translational conformations of nascent polypeptide chains. Methods in Enzymology 466, 565-588. [PDF]
Evans M.S., Sander I.M & Clark P.L. (2008) Co-translational folding promotes beta-helix formation and prevents aggregation in vivo. Journal of Molecular Biology 383, 683-692. [PDF]
Evans M.S., Ugrinov, K.G., Frese, M. & Clark P.L. (2005) Homogeneous stalled ribosome nascent chain complexes produced in vivo or in vitro. Nature Methods 2, 757-762. [PDF]
The distribution of rare codons in mRNA sequences
Our interest in co-translational folding of nascent polypeptide chains has led to a detailed exploration of how nascent chains enter the cellular milieu. While the translation rate for nascent chain synthesis by the ribosome is often reported as a bulk rate (typically 20 aa/sec in bacteria, or 4-6 aa/sec in eukaryotes), the local rate can vary by more than an order of magnitude. A wide array of forces can influence local translation rate, but we have focused on the effects of synonymous codon usage. Eighteen of the 20 common amino acids are encoded on mRNA sequences by more than one codon, and some of these codons are used more commonly than others. Rare codons are, on average, translated more slowly than common codons. We have shown that, contrary to expectations, rare codons are not randomly sprinkled across mRNA coding sequences, but instead often appear in large clusters. Moreover, the clusters are not randomly distributed, either, but occur more frequently at gene termini (5' and 3'). Large clusters have been identified in genes of all functional classes, and from diverse organisms, including all eukaryotes examined to date. And, these clusters can significantly reduce local translation rate. We are currently exploring the reasons for rare codon clustering.
Our online tool to calculate the rare codon distribution in your favorite gene is available here: Rare Codon Calculator.
Clarke T.F. IV & Clark P.L. (2010) Increased incidence of rare codon clusters at gene termini: Implications for function. BMC Genomics 11, 118. [PDF]
Clarke T.F. IV & Clark P.L. (2008) Rare codons cluster. PLoS ONE 3, e3412 doi:10.1371/journal.pone.0003412 . [PDF]