Crosslinking the 3′ End of a VLAT to RNA Polymerase: A Method to Elucidate Forward Hyper-Translocation
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On a typically strong E. coli promoter, as nascent RNA grows to 8-10 nt⎯accompanied by scrunching 6-8 bp of the enlarged transcription bubble⎯the initial DNA-RNAP contacts disrupt, upstream bubble rewinds, promoter escape occurs, and the elongation phase ensues. On some unique promoters, escape is accomplished only when the nascent RNA has grown to 16-19 nt in length. Such a promoter on the cusp of escape contains extremely high stress energy, which when utilized for upstream bubble rewinding and allowing promoter escape, can generate so much force as to propel the RNAP forward by several bases . We call this process forward hyper-translocation, during which the nascent RNA 3′-OH end is moved upstream into the RNA exit channel, out of register with the active site. The RNA molecule cannot be further elongated and is released as a VLAT. This mechanism can account for the GreB-resistant nature of VLAT synthesis. To prove that the VLAT production is the result of forward hyper-translocation, I conducted crosslinking experiments, incorporating a 4-thiouridine (S4U) residue as the 3′-most nucleotide of the VLAT RNA. As the result of UV irradiation, the 3′ terminus of the nascent RNA would form a covalent bond to the nearest protein residues of RNAP via the thiol group on S4U. This crosslinking approach was used successfully in locating the 3′ end of backtracked RNA molecules to the secondary channel of RNAP . If VLATs are in fact the products of forward hyper-translocation, the 3' end will now be located in the vicinity of the RNA exit channel. After performing several SDS-PAGE analysis of the crosslinking experiment, I have positive evidence showing that the 3′-most S4U residue of VLATs 18- and 19-nt in length do in fact crosslink to the β/β′ subunits of RNAP, lining the RNA exit channel. The crosslinking is UV-, RNAP-, and S4U-dependent. Techniques to enrich the crosslinked products were successful, including increasing UV exposure and DNA concentration and testing different UV irradiation wavelengths. To identify the exact amino acids/peptides the VLATs crosslinked to either β and/or β′, we need to reduce the size of the RNA-protein crosslinked complexes before subjecting it to MALDI-TOF mass spectrometric analysis. My first attempt of in-gel digestion of the crosslinked product with micrococcal nuclease was partially successful. Future studies will investigate the use of RNase T1 and trypsin digestion for generating crosslinked complexes whose size is appropriate for mass spec analysis.