The primary focus of the research will be on three very important features of prokaryotic transcription elongation: (i) processivity; (ii) the relationship between RNAP movement and nucleotide addition; (iii) fidelity and error correction. The work will be based on a new model for elongation recently proposed by Dr. Goldfarb, which constitutes a revision to the previously held """"""""inchworm"""""""" model. The main idea will be to use chemical crosslinking and protein chemistry to define the contacts made by different functional components or RNAP, including the front-end DNA binding site (DBS), the catalytic active center (AC), the RNA-DNA hybrid binding site (HBS), and the upstream RNA product binding site (RBS). In addition, the investigator proposes to dissect the basic nucleotide addition reaction into pre- and post-translocation states. During the past five years, a number of innovative techniques have been developed, primarily in the Goldfarb laboratory, which provide sophisticated tools for the investigation of structure-function relationships in elongation: (i) RNAP walking experiments are be done with His-tagged enzyme chelated to Ni-agarose beads, thus permitting the isolation of defined complexes at each step. (ii) Crosslinking probes can be incorporated into RNA at the 5' and 3' ends or within the body of the transcript, as well as in any position on the DNA template. These can then be used to demonstrate which parts of RNAP are contacted by the tagged nucleotide, and have also been used to demonstrate the formation and extent of the nascent RNA-DNA hybrid. (iii) Derivatized crosslinkable rifampicin compounds have been used to identify the relationship between the rif binding site and the transcription startsite. (iv) Fe++ -induced cleavage has helped identify sites in the enzyme that make up the active center (AC). A number of these methods have been used to characterize the active center, which appears to be made up of modules coming from distinct regions of both beta and beta'. The basic design of the proposed experiments will be to use specific radioactive crosslinking agents substituted at particular positions in the nascent RNA or in the DNA template, followed by cleavage of the crosslinked protein subunit with any of several different amino acid-specific cleavage reagents. Various crosslinkers with different crosslinking radii, or with the ability to be placed at different positions in nascent RNA, will be employed Many of these have been developed in the investigator's laboratory, or in collaboration with a Russian research group. Analysis of the crosslinked peptides will show which regions of the subunits are in contact with RNA or DNA at particular stages of the transcription cycle. Mapping will also be facilitated by introduction of histidine tags into beta and beta', substitution of methionines at well defined, non-essential sites to facilitate mapping with cyanogen bromide, and cleavage of beta and beta' split into sub-domains prior to mapping.
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