Structural basis for heterogeneous kinetics: Reengineering the hairpin ribozyme

The RNA cleavage reaction catalyzed by the hairpin ribozyme shows biphasic kinetics, and chase experiments show that the slow phase of the reaction results from reversible substrate binding to an inactive conformational isomer. To investigate the structural basis for the heterogeneous kinetics, we have developed an enzymatic RNA modification method that selectively traps substrate bound to the inactive conformer and allows the two forms of the ribozyme-substrate complex to be separated and analyzed by using both physical and kinetic strategies. The inactive form of the complex was trapped by the addition of T4 RNA ligase to a cleavage reaction, resulting in covalent linkage of the 5′ end of the substrate to the 3′ end of the ribozyme and in selective and quantitative ablation of the slow kinetic phase of the reaction. This result indicates that the inactive form of the ribozyme-substrate complex can adopt a conformation in which helices 2 and 3 are coaxially stacked, whereas the active form does not have access to this conformation, because of a sharp bend at the helical junction that presumably is stabilized by inter-domain tertiary contacts required for catalytic activity. These results were used to improve the activity of the hairpin ribozyme by designing new interfaces between the two domains, one containing a non-nucleotidic orthobenzene linkage and the other replacing the two-way junction with a three-way junction. Each of these modified ribozymes preferentially adopts the active conformation and displays improved catalytic efficiency.

(Table 1) Some structural characteristics of communities and detritus flux through the lower boundary of a given water column in different regions of the Kara Sea

Vertical fluxes of autochtonous detritus at different levels were estimated using the algorithm of structure-function analysis. The calculations are based on pelagic ecosystem parameters in the Kara Sea observed in September 1993 (temperature, primary production, biomass of phytoplankton, bacteria, protozoa, and zooplankton, trophic and size composition, etc.). At eight stations in different parts of the sea where sedimentation traps were set, the range of calculated fluxes of autochtonous detritus through the lower boundary of the water column was 13-90 mgC/m**2/day. The flux was much higher in the estuary of the Yenisey River (55-90 mgC/m**2/day) than in the northeastern regions (I8-50 mgC/m**2/day) and, especially, in the relatively deep southwestern part of the sea (13-35 mgC/m**2/day). The calculated fluxes of autochtonous detritus in shallow water regions (where conditions are variable and poorly known hydrologically and where outflow of allochtonous detritus is substantial) cannot be compared to data from sedimentation traps.

Guidebooks to the geology of Maryland.

Each part has special t.p. and separate paging.

Structural trends in the Illinois Basin,

Cover title.

Some stratigraphic, sedimentary, and structural relations of uranium deposits in the Salt Wash Sandstone : final report - April 1, 1952 to June 30, 1954 /

Under contract between the University of Utah and the U.S. Atomic Energy Commission, the writer has carried on a field and laboratory investigation of the uranium deposits in the Salt Wash sandstone of the Colorado Plateau.

Some structural features of the northern anthracite coal basin, Pennsylvania /

1 folded map in back pocket.

Paleochannel across Louden Anticline, Fayette County, Illinois : its relation to stratigraphic entrapment of petroleum in the Cypress sandstone /

Includes bibliographical references.

The occurrence of petroleum in Burma, and its technical exploitation /

Mode of access: Internet.

Popular oil geology,

"An elaboration of a number of popular lectures in the geology of oil and gas ... delivered during the past winter, in part at the Colorado school of mines and in part in the city of Denver, under the auspices of the Colorado state oil commission."--Pref.

Popular oil geology,

Mode of access: Internet.

Structural analysis of metamorphic tectonites

Mode of access: Internet.