Single Molecule Measurement of Peptide Elongation Rate During Synthesis of a Full-Length Protein

Abstract

The rate of translation of full-length proteins by the ribosome influences expression levels, folding and frameshifting. In order to study the factors that control translation rates, we use the expression of fast maturing Emerald Green Fluorescent Protein (EmGFP) by a reconsitituted E. coli cell-free translation system. Single-molecule TIRF microscopy allows monitoring of the appearance of single EmGFPs on the slide surface. Active translational complexes are docked to the microscope slide by a surface-immobilized antibody against an N-terminal extension of the translated protein. The appearance of fluorescent EmGFP spots identifies the end-point of full-length expression. In order to follow the translation rate in real time, the existing ribosomes and Phe-tRNAPhe in the cell-free mixture are replaced by fluorescent labeled Phe-tRNAPhe(Cy5.5) and L11(Cy3)-ribosomes, both of which are active in ensemble EmGFP expression assays. Individual Phe-tRNAPhes accommodated into the ribosome A-site during EmGFP translation are detected by single-molecule FRET pulses from Phe-tRNAPhe(Cy5.5) binding near the ribosomal protein L11(Cy3). Multiple accommodations of Phe-tRNAPhes on single ribosomes are observed during synthesis of EmGFP. The time intervals between two consecutive Phe-tRNAPhe accommodations can be assigned to particular sequence segments according to their temporal positions relative to two characteristic Phe-Phe doublets near the center of the EmGFP sequence. The assignments are validated by a decrease in translation rate when natural codons are replaced with codons pairing with rare isoacceptor tRNAs. This experimental platform will enable further testing of the control of translation rate by other rare codons, mRNA secondary structures, nascent polypeptide interactions with the ribosomal exit tunnel, and internal Shine-Dalgarno sequences. Supported by NIH Grant GM080376 and HFSP.