Poster abstracts

Poster number 35 submitted by Seth Lyon

Utilization of an RNase P-based assay for accurate measurement of 5'-modification in tRNAs

Seth Lyon (Chemistry and Biochemistry), Edward J. Behrman (Chemistry and Biochemistry), Venkat Gopalan (Chemistry and Biochemistry)

Abstract:
RNAs perform an array of functions in all life (e.g., catalysis, chromatin remodeling, structural scaffolds for large assemblies). Understanding this versatility of RNAs requires knowledge of their structure-function relationships. Probing RNA structure often requires spectroscopic methods, which in turn necessitates strategies for post-synthetic, site-specific incorporation of chemical probes into target RNAs. One method to achieve this goal is through in vitro transcription (IVT) of RNAs by T7 RNA polymerase and a GTP-initiating class III Φ6.5 promoter. In addition to GTP, T7 RNA polymerase can incorporate 5'-modified guanosine analogs during transcriptional priming. Because the nucleoside/nucleotide monophosphate guanosine analog cannot be used in elongation, it can only serve as the initiator. By using a 4-fold excess of the 5'-modified guanosine analog:GTP in the IVT, others have successfully biased the polymerase to initiate with the analog. We have now rigorously examined the extent of this bias with different RNAs and with 5'-deoxy-5'-azidoguanosine (Az-G) as the modifier. For small RNAs (5 nts), T7 RNA polymerase indeed generates mostly 5'-Az-G-modified RNAs (~75%). With tRNAs (150 nts), we determined that there is an unexpected maximum threshold (<50%) for the incorporation of Az-G into full-length transcripts even across a range of [Az-G]:[GTP] ratios. The use of RNase P, which catalyzes the cleavage of the 5'-leader in pre-tRNAs, was essential to obtain this accurate measurement. The ceiling that we observed for initiation of pre-tRNAs with 5'-modified guanosine analogs likely reflects a preponderance of 5'-Az-G-containing abortive transcripts, which are less likely when GTP is the initiator. Our work suggests that spectrophotometric (or other) methods previously used to determine the extent of incorporation overestimate the extent of 5'-modification by including both aborted and full-length RNAs. Our results should motivate use of T7 RNA polymerase mutants that have a decreased tendency for premature termination upon initiation with guanosine analogs.

Keywords: RNA-labeling