Protein synthesis rates during translation by the ribosome control many key cellular processes. Protein folding in living cells is inherently coupled to protein synthesis and chain elongation. There is considerable evidence that some nascent chains fold into their native structures in a cotranslational manner before release from the ribosome (1
). Furthermore, chaperones, such as GroEL, are estimated to assist folding of only ~5% of newly synthesized protein in growing Escherichia coli
). Translational elongation events such as pausing and frameshifting are linked to protein folding, export and expression.
Elongation rates have been measured using in vivo
and in vitro
translation assays in prokaryotic system. In vivo
, protein synthesis occurs at a rate of ~10–20 amino acids per second (3
), and dilution in extracts leads to slower rates in vitro
). Nascent chains are extruded from the ribosome through a long, narrow exit tunnel in the large subunit. This tunnel can accommodate ~22–26 amino acids, and constricts folding of higher order structure for the nascent chain (5
). A growing body of data suggests strong interplay of the nascent chain composition, the exit tunnel and elongation rates (5
The dynamics of full protein synthesis and cotranslational folding of nascent polypeptide remain unclear. Current bulk methods do not measure protein synthesis in real time; instead discrete time points are obtained to measure protein concentration biochemically, or enzymatic reporter systems are used, such as luciferase. Ensemble averaging in these bulk experiments blurs the detailed dynamic behavior of individual molecules. Single-molecule analysis of translation removes ensemble analysis and allows detailed examination of dynamics. However, single-molecule studies on protein synthesis to date have focused on individual kinetic steps, or overall protein synthesis in cells (7
); new methods are needed to couple ribosomal dynamics with protein synthesis and folding rates.
Here, we present a simple single-molecule system to monitor real-time translation and folding of proteins. Ribosomes are specifically immobilized on glass surfaces, and protein synthesis and folding is monitored in real time through synthesis and rapid maturation of a single green fluorescent protein (GFP) on the immobilized ribosome. However, GFP requires slow posttranslational modification of the folded protein to become fluorescent. Here, we use a fast-maturing mutant of GFP that provides improved time resolution for fluorescence. Nascent chains are stalled on immobilized ribosomes using the Secretion Monitor (SecM) arrest sequence, which lodges in the peptide exit tunnel and halts elongation. Thus, we can observe appearance of GFP fluorescence as a surrogate marker for coupled translation protein folding and chromophore maturation.