Steven M. Block - US grants

The overall goal of this proposed research is to investigate the control of peripheral vascular resistance in conscious neonatal lambs. The studies include 1) the definition of dose response relationships of phenylephrine (representative of alpha1- adrenergic stimulation), angiotensin II and vasopressin infused into the hind limbs of newborn lambs to femoral arterial blood flow in that limb; 2) the development of the model of local receptor blockade in the hind limb of the newborn lamb, focusing on the alpha1, vasopressin (V1) and angiotensin II receptors; 3) the determination of the hemodynamic and endocrine changes that occur in lambs in response to different degrees of blood loss; 4) the evaluation of the relative roles of receptors in the development of vascular resistance during hemorrhage by bleeding the animals during regional blockade. Chronically cannulated newborn lambs are chosen for the project because it is possible to study the cardiovascular and endocrine relationships in these animals in the absence of the physiologic changes brought about by surgery and anesthesia. The research will further knowledge on the control of peripheral vascular resistance by endocrine reflexes. Hypotension of various causes is a frequent clinical problem facing human newborns. Increased understanding of the adaptive mechanisms used by neonates in response to hypotension may prove important in the development of management strategies for the premature infant or the term infant with hypotension.

[unreadable] DESCRIPTION (provided by applicant): Motor proteins convert metabolic energy into force and displacement, generating movement in living organisms. The largest class of such proteins derives energy from the hydrolysis of ATP, and includes the myosin, dynein, and kinesin superfamilies. Despite over a century of study and the arsenal of chemical and physical approaches that has been tried, the molecular mechanism by which mechanoenzymes work remains obscure. Today, the mystery of motility is one of the outstanding problems in biology, with obvious implications in understanding the basis of motor-related disease. The advent of in vitro assays has, at last, allowed motor proteins to be studied in comparative isolation, using highly purified components interacting in defined geometries, in many cases down to the level of individual molecules. Among the motor proteins, the kinesin-microtubule system affords special advantages for study, because (1) kinesin and related proteins represent the smallest motors yet discovered, (2) processive motion can be generated by single kinesin motors, (3) the atomic structure of the kinesin motor domain bound to ADP has been solved, (4) recombinant kinesin derivatives and kinesin-related proteins can be isolated in functional form in both bacterial and eukaryotic expression systems, and (5) technology exists that can supply forces and measure displacements on the molecular scale, with high temporal and spatial resolution. Thanks, in part, to these advantages, great strides have recently been made towards establishing constraints on possible models for movement, vastly reducing the constellation of mechanisms to consider. The long-term goal of this research is to dev-elop a quantitative understanding of how kinesin proteins function, based on detailed molecular physiology combined with biochemical and biostructurai data. Specific aims include measurement of the speeds, forces, displacements, cycle timing, ATP coupling, head-head interactions, and other properties of kinesin, kinesin-related proteins, and genetically-engineered derivatives thereof. For this purpose, advanced instrumentation based on optical trapping ('optical tweezers') and optical nanometry has been developed, and will be used in experiments conducted at the single-molecule level.

This application describes a research program to characterize the process of transcription by E. Coli RNA polymerase molecules at the single molecule level by means of novel biophysical methods of optical trapping and nanometer-level position sensing. The ultimate objective of this proposal is to investigate the mechanochemical properties of this processive enzyme, to gain an understanding of how the DNA-direct RNA synthesis is coupled to translocation along the DNA template. The specific aims of this proposal are: 1) To determine the force- velocity relationship for RNAP and characterize themechanochemical properties of this motor protein. These studies will investigate the mechanochemical behavior of this enzyme spanning the full range of external load forces; i.e., below, at and above the stall force of the motor. Similarly, the force- velocity relationship of the enzyme working in the presence of an external load applied in the direction of the motion will be characterized. 2) Investigate transcriptional pausing, stalling and arrest by RNAP and its relationship to the nature of the DNA template. These experiments will investigate whether the arrest of the enzyme is a probabilistic or a deterministic phenomenon, whether it is dictated by the sequence of the template, and if reversible and irreversible arrests are influenced by the present and the magnitude of the external load. The sequence effects onpausing and stalling will be studied on complex sequences, on direct repeat templates, and on homopolymers. Attempts will be also made to observe, directly, slippage under the external load. Finally, these studies will investigate what is the effect of transcript cleavage factors such as GreA and GreB on pausing and stalling. 3) Study the fluctuation behavior of the dynamics of individual transcribing enzymes. A variance analysis will be applied to obtain information about the number and duration of the rate-limiging steps in the enzyme reaction, as a way to get an insight into the molecular mechanism responsible for the mechanochemical coupling in RNAP. 4) Studies of the fine structure of the translocation of transcribing single RNAP molecules. These studies will be designed to I) investigate the effect of torsional strain during transcription, ii) obtain direct evidence of discontinuous or "inch worming" movement of the polymerase along the template, particularly during the early phases of transcription, and iii) attempt to resolve the process of transcription to single base pair resolution.

Motor proteins convert metabolic energy into force and displacement, generating movement in living organisms. The largest class of such proteins derives energy from the hydrolysis of ATP, and includes the myosin, dynein, and kinesin superfamilies. Despite over a century of study and the arsenal of chemical and physical approaches that has been tried, the molecular mechanism by which mechanoenzymes actually work remains obscure. Today, the mystery of force production is one of the outstanding problems in biology, with obvious implications in understanding the basis of motor-related disease. The advent of in vitro motility assays has, at last allowed motor proteins to be studied in cimparative isolation, using purified components interacting in defined geometries, in many cases down the level of individual molecules. Among the motor proteins, the kinesin-microtuble system affords certain advantages for stud, because (1) kinesin and related proteins represent the smallest motors yet discovered, (2) processive motion can be generated by single kinesin motors, (3) the atomic structure of the kinesin motor domain has been solved, (4) recombinant kinesin derivatives and kinesin-related proteins can be isolated in functional form, in both bacterial and eukaryotic expression systems, and (5) technology exists that can supply forces and measure displacements on the molecular scale, with high temporal and spatial resolution. Thanks, in part, to these advantages, great strides have recently been made towards establishing constraints on possible models for movement, vastly reducing the constellation of mechanisms to consider. The long-term goal of this research is to develop a quantitative understanding of kinesin protein function, based on detailed molecular physiology, combined with biochemical and biostructural data. Specific aims include measurement of the speeds, forces, displacements, cycle timing, ATP coupling, head-head interactions, and other properties of kinesin, kinesin-related proteins, and genetically-engineered derivatives thereof. For this purpose, advanced instrumentation based on optical trapping (optical tweezers) and optical nanometry has been developed, and will be used in experiments at the single molecule level.

The Central Dogma of biology[unreadable]whereby DMA is replicated, transcribed, and ultimately translated into protein[unreadable]is carried out by a handful of important enzymes. These enzymes, which include polymerases, helicases, nucleases, and ribosomes, constitute a core group of sophisticated, biomolecular machines. A detailed understanding of such machines is key to any understanding of life itself, and by extension, to the treatment of human disease. Furthermore, progress towards revealing the molecular mechanisms that power Nature's own molecular-scale machines is directly informing much of our current work at the forefront of nanotechnology, which promises to harness the power of nanoscale devices for the betterment of the human condition. Put simply, we need to know how these machines work if we're to fix them[unreadable]or to copy them. A property shared by many nucleic acid enzymes is that they function as processive motors: once bound to their DNA template, they carry out repeated enzymatic cycles, often moving long distances before detaching. This motion is accompanied by the production of force, and requires a continuous input of chemical energy, usually in the form of nucleoside triphosphates. In contrast to classical mechanoenzymes like myosin (which moves muscles) or kinesin (which transports organelles in cells), the motor-like properties of nucleic acid enzymes are continually modulated by the changing information found in the DNA template, yielding a much richer dynamic behavior. Although high-resolution structural data have become available for many important nucleic acid enzymes, comparatively little is understood about the underlying molecular mechanisms. Recently, work on molecular motors has been revolutionized by the ability to measure force and displace- ment at the level of single molecules, using a new generation of biophysical instrumentation, including laser- based optical traps, scanning force microscopy, and advanced fluorescence techniques that can score single photons. Single-molecule studies hold great promise because they supply unique information[unreadable]particularly about the distribution and heterogeneity of enzymatic properties[unreadable]that's been largely inaccessible using traditional biochemical or genetic approaches. An assay developed by my group has allowed us to study gene transcription by E. coli RNA polymerase (RNAP) in real time at the level of individual molecules using optical traps. Our prior work with this system has raised specific questions about the elongation mechanism, the load-dependence, the DNA sequence specificity, pausing &stalling behavior, enzyme microstates, enzyme regulation, repair mechanisms, etc.,that we're now in an excellent position to address through continuing study. Single-molecule techniques may even become sensitive enough to measure the size of the individual steps taken by RNAP, which are expected to correspond directly to the spacing of individual bases along the DNA (3.4A). A direct demonstration that RNAP advances in single basepair increments would allow us to rule out a competing theory that it moves by a so-called "inchworm" mechanism.

The Central Dogma of Biology - whereby genetic information gets replicated, transcribed, and translated into proteins-is carried out by a handful of critical enzymes that include polymerases, helicases, and ribosomes. Together, these enzymes constitute a core group of sophisticated, biomolecular machines Understanding their function holds a key to understanding life, and by extension, to the treatment of human disease. Hard-won knowledge about biomolecules is informing work at the forefront of nanoscience,: which hopes to harness tiny devices to better the human condition. Put simply, we need to know how Nature's machines work if we're ever to fix them or to emulate them. A property shared by many nucleic acid-based enzymes is that they function as molecular motors: once bound to DNA or RNA, they undergo repeated cycles, often traveling considerable distances. This motion is accompanied by force production and requires chemical energy. In contrast tomechanoenzymes like myosin, the properties of nucleic acid-based motors axe modulated by an ever-changing template underfoot, yielding rich behavior. Although structural data are available for many such enzymes, comparatively little is known about their molecular mechanisms Recently, biophysical studies have been revolutionized by the ability to measure forces and displacements at the level of single molecules, using techniques that include optical traps, nanometry, and fluorescence Single-molecule approaches supply critical information that has been hitherto inaccessible by traditional techniques. Previously, my group succeeded in developing optical trapping instrumentation that?s able to register displacements down to the atomic level (~1 A). Consequently, we can record from bacterial RNA polymerase (RNAP) molecules as these step from base to base along DNA. Improved instrumental stability now allows us to reconstruct energy landscapes for folding transitions in nucleic acids that form complex structures (riboswitches, ribozymes, etc.). We propose to continue our single-molecule work on transcription by RNAP. We also plan to use single-molecule assays to address unsolved problems of co-transcriptional folding and gene regulation, and to better understand the sequence elements that regulate transcriptional elongation and termination {e.g., riboswitches). A closely related assay will allow us to study the initiation of translation by ribosomes in eukaryotes, along with the RNA sequence elements that modulate that process. Finally, we will pursue a successful single-molecule assay we developed for transcription by Pol II (RNAPII), the eukaryotic analog of RNAP.