My lab conducts high-resolution, single-molecule studies of the molecular mechanisms of ribosome functions and gene expression control.
Translation, the process of protein synthesis by ribosome decoding of the genetic codes on messenger RNA, is one of the most fundamental biological processes in all three kingdoms of life. Translation is closely regulated in the cell to coordinate with other cellular events and to adapt to environmental stress. Abnormality in translational control of gene expression in humans has been implicated in many diseases, such as cancer, metabolic diseases (for example, obesity and diabetes), and viral infection. Drugs have been developed to: (i) treat tumors by down-regulating translation; (ii) treat viral infection by altering frame-shifting efficiency; (iii) treat genetic disorders by promoting read-through of nonsense mutations; and (iv) treat microbial infection by inhibiting the prokaryotic ribosome function. Understanding the mechanism of translational control of gene expression is of great significance for developing better drugs and fighting human diseases.
Diverse mechanisms have been discovered for translational control of gene expression, but our understanding is still greatly lacking in many areas as a result of the inherent complexity of the translation system. Specifically, translational control is a very fast, dynamic process involving many protein and RNA factors. This imposes great technical challenges for monitoring translational control in real time on the molecular level, which has a minimal requirement of simultaneous millisecond time resolution and nanometer spatial resolution. Such requirement is out of the range of traditional biochemical and biophysical techniques. The recent developments in single-molecule techniques filled the gap and opened the door to elevate our understanding of translational control to a new level. Examples of two types of single-molecule techniques are shown below. The goal of our lab is to unravel the fundamental molecular mechanisms of translational control of gene expression by integrating advanced single-molecule optical microscopy, biophysical and biochemical techniques, and quantitative modeling.
Example application of optical tweezers to study translational control. In this experimental set-up (a), the two ends of the mRNA are attached to two micron-sized polystyrene beads, which allow the laser trap to measure the end-to-end distance of the mRNA with subnanometer spatial resolution, the force on the mRNA with sub-piconewton resolution, and the dynamics of translation with submillisecond resolution. When ribosome translates the gene encoded on this mRNA, the codon-by-codon translation activity will be accompanied by a step-wise increase of the mRNA end-to-end distance over time (b). Optical tweezers offer the best combined spatial and temporal resolutions compared with other techniques. The ribosome-mRNA complex is immersed in the translation mixture, which contains all the necessary factors (such as tRNAs, EF-G, and EF-Tu) to allow translation. The effects of regulatory elements on the rate of translation can be readily measured, by changing their concentrations in the translation mixture.
Example application of single-molecule fluorescence resonance energy transfer (smFRET) to study translational control. In this experimental set-up (a), the 30S and 50S subunits of the ribosome are labeled with a donor dye (green star) and an acceptor dye (red star), respectively. During translation, the ribosome undergoes frequent inter-subunit conformational changes, which will be reflected by the change in the donor and acceptor emission intensities (b). smFRET usually allows a spatial resolution of a few nanometers, and a temporal resolution of several tens of milliseconds. Even though its resolutions are inferior to optical tweezers, smFRET is a very versatile technique. Besides labeling the subunits of the ribosome, other factors (tRNAs, mRNA, translation factors, regulatory proteins, non-coding RNAs, etc.) can also be labeled, and studies by smFRET. smFRET can be readily combined with multicolor fluorescence imaging to monitor the interplay of all the labeled components in real time. smFRET, and fluorescence techniques in general, also bear great potential for measurements in living cells.