A New Paradigm
A new paradigm exists for understanding how cells function. Scientists are recognizing that the cell is a highly integrated biological factory with a modular architecture. Each modular unit acts as a molecular machine. These machines have highly specialized functions and are large assemblies of proteins and nucleic acids. They range in size from about 10 - 150 nanometers (10-9 m) and provide environments in which chemical species can interact in a highly specific fashion. Molecular machines also function as mechano-chemical energy transducers, converting chemical free energy into mechanical energy for cellular processes. They operate cyclically, and can reset themselves.
With the genetic information gained from the U.S. Human Genome Project and DOE's Microbial Genome Program, scientists now have the raw information with which to observe, manipulate, characterize and, ultimately, replicate these large protein assemblies. Using conventional and newly developed microscopy techniques, PBD researchers, through an initiative called Microscopies of Molecular Machines (M3), are creating a toolkit for probing the inner workings of these molecular machines.
Tools for Investigating Nanometer-scale Machines
Physical Biosciences investigators, in concert with other Berkeley Lab investigators, are developing novel, complementary combinations of established and newly developed microscopy tools for macromolecular analysis. Four of these tools are: atomic force microscopy (AFM), cryo-electron microscopy, single-molecule fluorescence microscopy (SFM), and optical tweezers microscopy. All of these methods rely on single molecule analysis and provide complementary, structural, and dynamic biological information.
AFM is a descendent of scanning tunneling microscopy (STM). Images are generated by recording the deflection of a very sharp tip as it is dragged over a sample. In this way, an atom by atom picture of the surface is taken.
Cryo-electron microscopy is a relatively new technique that ensures preservation of a biological sample and accuracy of the images, compared to standard electron microscopy. Samples are prepared by rapidly freezing them, and are then preserved in an aqueous-like vacuum environment. There are few artifacts, staining is not required, and radiation damage is minimal.
Single molecule fluorescence (SMF) is a new type of biological imaging technique where light emitted from individual fluorophores (e.g. quantum dots) is captured and used to investigate the conformational dynamics of proteins and nuclic acids.
Optical tweezers microscopy is a relatively new tool (~ late 80's and early 90's) that permits molecule-by-molecule measurement and manipulation of a sample. Through the use of a highly controlled laser beam, the mechanical properties of DNA are measured by grabbing the DNA at both ends and pulling it, thus revealing its resistance to stretching.
Currently, we are refining these technolgies (e.g. by improving their temporal or spatial resolution), as well as developing entirely new "hybrid" instruments by combining AFM with SMF, and optical tweezers with SMF. The goal of the latter effort (tentatively called "Initiative for Advanced Instrumentation") is to develop and construct hybrid instruments that will allow scientists to mechanically manipulate molecular machines (using AFM or optical tweezers) while simultaneously watching the machine respond to the external perturbation using SMF. Such instrumentation will not only benefit efforts to understand biological machines, but also assist in the construction of entirely new machines and devices from scratch, the focus of the Molecular Foundry program at Berkeley.
Research Focus - Cellular Processes Under Study
With the use of these and other tools, M3 is initially focused on fundamental cellular processes, like DNA Replication and Repair, DNA Transcription, and Synaptic Transmission and Plasticity. Also, we are interested to understand the physics of how molecular motors work, e.g. the energetics and kinetics of mechanochemical energy conversion.
DNA replication, repair, and transcription are the essential processes through which the cell preserves and utilizes its genetic information. DNA replication ensures the passage of information contained in the DNA from one generation to the next. Transcription governs the regulation of gene expression and therefore functional identity. The study of the complex structural dynamics underlying gene expression and DNA replication and repair by these new microscopies will contribute to the DOE's effort to understand the effects of radiation on and chemical damage to biological systems and human health. We have recently begun to study the effect of DNA lesions on DNA transcription by RNA polymerases. In these studies, we are using optical tweezers to watch how individual polymerase molecules respond to DNA damage such as nicks and crosslinks.
Synaptic transmission and plasticity examines the synaptic machinery that is at the heart of the nervous system's communication network and the site through which neural connections are made. The molecular machinery of the synapse contains protein complexes that are responsible for memory and learning. The microscopies used by M3 allow one to study these proteins in their native environment and measure their interactions within the synapses.
A Look at Transcription Regulation Using the M3 Toolkit
Using cryo-electron microscopy and AFM, M3 scientists recently obtained the structures of human TFIID and the TFIID-IIA-IIB complex (a horseshoe-shaped structure) to a 35-angstrom resolution. TFIID (a protein polymer) is an essential component of the RNA polymerase II machinery. TFIID binds to the DNA promoter of every gene and recruits RNA polymerases, enzymes required in the synthesis of RNA using DNA or RNA as a template.