Translation by Single Ribosomes Through mRNA Secondary Structures

Abstract

During protein synthesis, the ribosome moves along a single mRNA strand and translates the genetic code into polypeptide sequences. Strong mRNA secondary structures, which have to be unfolded in order to be translated, can slow or even halt protein synthesis. Pseudo-knots (PKs), mRNA secondary structures containing at least two stem-loops, are essential elements in programmed ribosomal frameshifting, for instance, during translation of some viral mRNAs. Here we employ single molecule fluorescence resonance energy transfer (smFRET) to determine reaction rates for specific polypeptide elongation cycles as the ribosome encounters a minimal viral PK structure during mRNA translation. An elongation cycle may be divided into three steps: 1) aminoacyl-tRNA binding to the A-site, 2) translocation of the bound mRNA and tRNAs from the A- and P- sites to the P- and E-sites, and 3) dissociation of deacylated tRNA from the E-site. We use FRET interactions between i) adjacent ribosome-bound tRNAs, ii) aminoacyl-tRNA and ribosomal protein L11 at the A-site, and iii) deacylated tRNA and ribosomal protein L1 at the E-site, to determine the rates of steps 1) - 3) during elongation cycles of ribosomes programmed with mRNAs either containing or lacking a PK. We find that, whereas the presence of a PK has little or no effect on the rates of steps 1) and 2), it strongly decreases (∼2.5-fold) the rate of step 3. Thus, somewhat surprisingly, step 3) appears to be more strongly coupled to the unfolding of PK structure than step 2). In contrast, preliminary results indicate that stem-loop structures can decrease the rates of both steps 2) and 3). Supported by NIH grant R01GM080376.