Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The force produced by a single molecule of Escherichia coli RNA polymerase during transcription was measured optically. Polymerase immobilized on a surface was used to transcribe a DNA template attached to a polystyrene bead 0.5 micrometer in diameter. The bead position was measured by interferometry while a force opposing translocation of the polymerase along the DNA was applied with an optical trap. At saturating nucleoside triphosphate concentrations, polymerase molecules stalled reversibly at a mean applied force estimated to be 14 piconewtons. This force is substantially larger than those measured for the cytoskeletal motors kinesin and myosin and exceeds mechanical loads that are estimated to oppose transcriptional elongation in vivo. The data are consistent with efficient conversion of the free energy liberated by RNA synthesis into mechanical work.
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PMID:Transcription against an applied force. 750 62

To examine the possible role of kinesin in pigment granule migration in the retinal pigment epithelium (RPE) of teleosts, we investigated the expression and distribution of kinesin heavy chain (KHC) in RPE. Blots of fish RPE lysates probed with two well-characterized antibodies to KHC (H2 and HD) displayed a prominent band at 120 kD. A third KHC antibody (SUK4) recognized a band at 118 kD. The 118 kD band was also occasionally present in blots probed with H2, suggesting the presence of two KHC isoforms in teleost RPE. Reverse transcriptase-polymerase chain reaction (RT-PCR) of mRNA from RPE using primers homologous to conserved regions of the KHC motor domain resulted in the identification of two putative KHC genes (FKIF1 and FKIF5) based on partial amino acid sequences. Previous studies had demonstrated a requirement for microtubules in pigment granule aggregation in RPE. In addition, the reported microtubule polarity orientation in RPE apical projections is consistent with a role for kinesin in pigment granule aggregation. Immunofluorescent localization of KHC in isolated RPE cells using H2 revealed a mottled distribution over the entire cell body, with no detectable selective association with pigment granules, even in cells fixed while aggregating pigment granules. Microinjected KHC antibodies had no effect on pigment granule aggregation or dispersion, although each of the three antibodies has been shown to block kinesin function in other systems. Thus we found no evidence for KHC function in RPE pigment granule aggregation. However, the two KHC isoforms may participate in other microtubule-dependent processes in RPE.
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PMID:Expression of kinesin heavy chain isoforms in retinal pigment epithelial cells. 755 3

Novel techniques are revealing the movements and forces associated with single interactions of motor proteins, such as myosin and kinesin, and also of processive enzymes, such as RNA polymerase.
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PMID:Molecular motors: single-molecule mechanics. 872 41

Novel techniques are revealing the movements and forces associated with single interactions of motor proteins, such as myosin and kinesin, and also of processive enzymes, such as RNA polymerase.
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PMID:Single-molecule mechanics. 880 54

A general mechanism for polymerase translocation is elaborated. The central feature of this mechanism is that a rapid translocational equilibrium is established after each cycle of nucleoside monophosphate incorporation such that the polymerase distributes itself by diffusional sliding between all accessible positions on the template with relative occupancy determined by relative free energy. While alternative models for translocation have not been fully developed, much of the language currently used to describe this step suggests an active mechanism coupled to conformational transitions in the polymerase. For example, a recent study of force generation by Escherichia coli RNA polymerase during transcription suggests that it is a mechanoenzyme analogous to kinesin of myosin motor proteins. While the proposed mechanism does not rule out conformational transitions during polymerase translocation, it suggests that they may be unnecessary and that translocation can be explained in terms of the affinity of the active site for nucleoside triphosphate and the relative free energies of the polymerase bound at different positions on the template. This mechanism makes specific predictions which are borne out experimentally with polymerases as distinct as E. coli DNAP I, phage T7 RNAP, and E. coli RNAP.
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PMID:A model for the mechanism of polymerase translocation. 899 20

Cells employ a variety of linear motors, such as myosin, kinesin and RNA polymerase, which move along and exert force on a filamentous structure. But only one rotary motor has been investigated in detail, the bacterial flagellum (a complex of about 100 protein molecules). We now show that a single molecule of F1-ATPase acts as a rotary motor, the smallest known, by direct observation of its motion. A central rotor of radius approximately 1 nm, formed by its gamma-subunit, turns in a stator barrel of radius approximately 5nm formed by three alpha- and three beta-subunits. F1-ATPase, together with the membrane-embedded proton-conducting unit F0, forms the H+-ATP synthase that reversibly couples transmembrane proton flow to ATP synthesis/hydrolysis in respiring and photosynthetic cells. It has been suggested that the gamma-subunit of F1-ATPase rotates within the alphabeta-hexamer, a conjecture supported by structural, biochemical and spectroscopic studies. We attached a fluorescent actin filament to the gamma-subunit as a marker, which enabled us to observe this motion directly. In the presence of ATP, the filament rotated for more than 100 revolutions in an anticlockwise direction when viewed from the 'membrane' side. The rotary torque produced reached more than 40 pN nm(-1) under high load.
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PMID:Direct observation of the rotation of F1-ATPase. 906 74

RNA polymerase (RNAP) moves along DNA while carrying out transcription, acting as a molecular motor. Transcriptional velocities for single molecules of Escherichia coli RNAP were measured as progressively larger forces were applied by a feedback-controlled optical trap. The shapes of RNAP force-velocity curves are distinct from those of the motor enzymes myosin or kinesin, and indicate that biochemical steps limiting transcription rates at low loads do not generate movement. Modeling the data suggests that high loads may halt RNAP by promoting a structural change which moves all or part of the enzyme backwards through a comparatively large distance, corresponding to 5 to 10 base pairs. This contrasts with previous models that assumed force acts directly upon a single-base translocation step.
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PMID:Force and velocity measured for single molecules of RNA polymerase. 979 53

The mechanical manipulation of single biological molecules is stimulating new and exciting research in many fields of study, including molecular motor mechanics, biopolymer properties, protein unfolding, receptor-ligand interactions, and more. Some recent highlights include the elucidation of the coupling ratios of myosin and kinesin, the demonstration of oscillatory forces in dynein arms, the determination of the force-velocity relation of RNA polymerase, and the direct mechanical observation of unfolding of single domains of titin and tenascin.
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PMID:Manipulation of single molecules in biology. 1004 11

Animal- and human-made motors vary widely in size and shape, are constructed of vastly different materials, use different mechanisms, and produce an enormous range of mass-specific power. Despite these differences, there is remarkable consistency in the maximum net force produced by broad classes of animal- and human-made motors. Motors that use force production to accomplish steady translational motion of a load (myosin, kinesin, dynein, and RNA polymerase molecules, muscle cells, whole muscles, winches, linear actuators, and rockets) have maximal force outputs that scale as the two-thirds power of mass, i.e., with cross-sectional area. Motors that use cyclical motion to generate force and are more subject to multiaxial stress and vibration have maximal force outputs that scale as a single isometric function of motor mass with mass-specific net force output averaging 57 N x kg(-1) (SD = 14). Examples of this class of motors includes flying birds, bats, and insects, swimming fish, various taxa of running animals, piston engines, electric motors, and all types of jets. Dependence of force production and stress resistance on cross-sectional area is well known, but the isometric scaling and common upper limit of mass-specific force production by cyclical motion motors has not been recognized previously and is not explained by an existing body of theory. Remarkably, this finding indicates that most of the motors used by humans and animals for transportation have a common upper limit of mass-specific net force output that is independent of materials and mechanisms.
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PMID:Molecules, muscles, and machines: universal performance characteristics of motors. 1191 97

A common feature in the maturation of linear dsDNA viruses is that the lengthy viral genome is translocated with remarkable velocity into a limited space within a preformed protein shell using ATP as motor energy. Most biomotors, such as myosin, kinesin, DNA-helicase, and RNA polymerase, contain one ATP-binding component that acts processively. An examination of the well-studied dsDNA viruses reveals that DNA packaging motors involve two nonstructural components. Which component of the motor is the integrated processive factor to turn the motor has not been identified. In bacterial virus phi 29, these two components consist of a gp16 protein and an RNA molecule called pRNA. We have previously predicted and recently confirmed that gp16 binds ATP. It is generally believed that gp16 serves as an ATP-binding and processive component to drive the motor. In this article, phi 29 DNA-packaging intermediates were purified in quantity and examined to differentiate the role between gp16 and pRNA. It was found that the pRNA hexamer is an integral motor component, while gp16 is not stably bound. Only one pRNA hexamer, but multiple copies of gp16, were needed to accomplish DNA packaging. pRNA functions continuously during the entire DNA translocation process, suggesting that pRNA is a vital part of the DNA packaging motor.
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PMID:Only one pRNA hexamer but multiple copies of the DNA-packaging protein gp16 are needed for the motor to package bacterial virus phi29 genomic DNA. 1272 31


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