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During protein synthesis, the ribosome translates nucleotide triplets in single-stranded mRNA into polypeptide sequences. Strong downstream mRNA secondary structures, which must be unfolded for translation, can slow or even halt protein synthesis. Here we used single-molecule fluorescence resonance energy transfer to determine reaction rates for specific(More)
We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations(More)
During protein synthesis, deacylated transfer RNAs leave the ribosome via an exit (E) site after mRNA translocation. How the ribosome regulates tRNA dissociation and whether functional linkages between the aminoacyl (A) and E sites modulate the dynamics of protein synthesis have long been debated. Using single molecule fluorescence resonance energy transfer(More)
Metal-enhanced fluorescence (MEF) increased total photon emission of Cy3- and Cy5-labeled ribosomal initiation complexes near 50 nm silver particles 4- and 5.5-fold, respectively. Fluorescence intensity fluctuations above shot noise, at 0.1-5 Hz, were greater on silver particles. Overall signal-to-noise ratio was similar or slightly improved near the(More)
EF4 (LepA), a strongly conserved protein, is important for bacterial growth and functional protein biosynthesis under certain conditions and is quite similar structurally to the translocase EF-G. The elongation cycle in protein synthesis is characterized by ribosome oscillation between pretranslocation (PRE) and posttranslocation (POST) complexes. Here,(More)
Hybridization of nucleic acids with secondary structure is involved in many biological processes and technological applications. To gain more insight into its mechanism, we have investigated the kinetics of DNA hybridization/denaturation via fluorescence resonance energy transfer (FRET) on perfectly matched and single-base-mismatched DNA strands. DNA(More)
Combining gold nanoparticles (GNPs) as fluorescence quencher and aptamer as probe, we have developed protein biosensors by using DNA-modified GNPs. We examined how the experimental design, such as the type of interaction between DNA strands and GNPs, temperature, and microenvironment of aptamer, influences the recognition ability of the biosensor. Under our(More)
Conventional single nucleotide polymorphism (SNP) assays, which based their detection on the stringency or temperature of the washing buffers, have encountered difficulties to distinguish a single base pair mismatch from a perfect match. In this study, scanning potential hairpin denaturation (SPHD) has been developed to detect SNP in a sensitive and(More)
Recordings from single molecule experiments can be aggregated to determine average kinetic properties of the system under observation. The kinetics after a synchronized reaction step can be interpreted using all of the standard tools developed for ensemble perturbation experiments. The kinetics leading up to a synchronized event, determined by the lifetimes(More)
We present proof-of-concept in vitro results demonstrating the feasibility of using single molecule fluorescence resonance energy transfer (smFRET) measurements to distinguish, in real time, between individual ribosomes programmed with several different, short mRNAs. For these measurements we use either the FRET signal generated between two tRNAs labeled(More)