Protein synthesis by single ribosomes

Abstract

The ribosome is universally responsible for synthesizing proteins by translating the genetic code transcribed in mRNA into an amino acid sequence. Ribosomes use cellular accessory proteins, soluble transfer RNAs, and metabolic energy to accomplish the initiation, elongation, and termination of peptide synthesis. In translocating processively along the mRNA template during the elongation cycle, ribosomes act as supramolecular motors. Here we demonstrate that ribosomes adsorbed on a surface, as for mechanical or spectroscopic studies, are capable of polypeptide synthesis and that tethered particle analysis of fluorescent beads connected to ribosomes via polyuridylic acid can be used to estimate the rate of polyphenylalanine synthesis by individual ribosomes. This work opens the way for application of biophysical techniques, originally developed for the classical motor proteins, to the understanding of protein biosynthesis.

Stochastic kinetics of ribosomes: Single motor properties and collective behavior

“…DWELL TIME DISTRIBUTION FOR A SINGLE RIBOSOME: MOST GENERAL CASE Because of recent improvements in experimental techniques, it has become possible to image and manipulate single ribosomes [6,15,16,17,18,19,20,21]. In the recent experiments on single-ribosome manipulation [6], the distribution of the dwell times of a single ribosome at a codon was measured.…”

“…The force-velocity relation V (F ) (i.e., the variation of the average velocity V of a motor with increasing load force F ) is one of the most important characteristics of a molecular motor. Inspired by the recent progress in the single-ribosome imaging and manipulation techniques [6,15,16,17,18,19,20,21], we extend the formula for t −1 appropriately to derive V (F ) for single ribosomes. The smallest load force which is just adequate to stall a molecular motor on its track is called the stall force F s .…”

Volume-Exclusion Effects in Tethered-Particle Experiments: Bead Size Matters

“…For many experiments [3][4][5][7][8][9][10]] the excursion number is such that the second term in Eq. (11) (6)].…”

“…Thus, the observed motion of the bead serves as a reporter of the underlying, invisible, macromolecular motion. This technique has been used in a variety of settings, e.g., the examination of nanometerscale motions of motors like kinesin [1] or RNA polymerase [2,3], protein synthesis by ribosomes [4], exonuclease translocation on DNA [5,6], protein mediated deformation [7] and loop formation [8] in DNA, DNA hybridization [9], and DNA motion [10,11]. The main goal of this Letter is to show how the proximity of the reporter bead to the surface affects the interpretation of the reported data and can even alter the conformation of the macromolecule of interest.…”

“…The key measurable associated with current TPM experiments is the position of the bead itself. That is, the output of the experiment is a record of the positions of the bead on successive video frames [4,10,11,14]. Having revealed that the confinement of the bead subjects the molecule to an entropic force, we now determine how this confinement influences bead motion.…”