RNA-Binding to Archaeal RNA Polymerase Subunits F/E: A DEER and FRET Study

摘要

DNA-dependent RNA polymerases (RNAPs) are complex mul-ntisubunit enzymes that facilitate transcription of all cellular genomes.nThe archaeal RNAP and eukaryotic RNAP are closely related innterms of subunit composition, ternary architecture, and requirementsnfor basal transcription factors. Recently hyperthermophilic archaealnRNAPs have emerged as versatile model systems for the lessntractable mesophilic eukaryotic RNAPII.n1,2nDuring transcriptionnRNAP interacts with double-stranded DNA, a 9 base pair DNARNA hybrid, and the nascent RNA chain. Biochemical studies havenshown that the eukaryotic RNAPII subunits RPB4/7 and theirnarchaeal homologues F/E bind the emerging RNA transcript in annonsequence-specific manner over a range of approximately 20nnucleotides.n3,4nThis interaction stimulates the processivity of thenarchaeal RNAP.n5nDuring transcription initiation, prior to RNAnsynthesis, F/E facilitates interaction with the basal transcriptionnfactor TFE and stimulates DNA-strand separation.n6,7nThe molecularnmechanisms that underlie the interplay between TFE, F/E and RNAnduring transcription are poorly understood but are likely to involvendynamic conformational changes of the RNAP nucleic acidncomplex.n8nSubunits F/E form a stable heterodimern9nthat associatesnstably with the RNAP core10n(Figure 1a). Structural and biophysicalnstudies have provided insights into the topology of downstreamnand upstream DNA and the DNA/RNA hybrid in the context ofnthe elongating RNAP.n11,12nHowever, the nascent transcript and itsninteraction with F/E-like subunits have not yet been captured.