Closing and opening of the RNA polymerase “trigger loop” in solution
Creators: name: Lin, Miaoxin
Publisher: name: No Publisher Supplied
Year: 2017

description: Previous crystal structures of transcription elongation complexes (TEC) suggested that each nucleotide-addition cycle is coupled to a closing/opening conformational cycle of the RNA polymerase (RNAP) active center, accomplished by folding (closing) of a structural element termed "trigger loop" (TL) upon binding of the incoming NTP, followed by unfolding (opening) of the RNAP TL upon formation of the phosphodiester bond. However, direct evidence for TL closing and opening in solution is lacking.   In this work, we have directly detected TL closing/opening in solution by measuring ensemble fluorescence resonance energy transfer (FRET) between two fluorescent probes in a labelled TEC without and with a bound NTP. As the FRET donor, a coumarin-derived fluorescent amino acid L-(7-hydroxycoumarin-4-yl)-ethylglycine (Cou) was incorporated by unnatural amino acid mutagenesis into TL. As the FRET acceptor, fluorescein was chemically coupled to each of eleven reference sites in DNA template strand. FRET measurements showed that in solution TL is open in the absence of bound NTP and closes after NTP binding.   In addition, we have analyzed the effects of substrate identities on TL conformation in solution by measuring ensemble FRET of labelled TEC with one of a series of NTPs or NTP analogues. The results indicated that: (i) TL closing depends on base complementarity; (ii) TL closing depends on tri-/di- /mono-phosphate identity; (iii) TL closing partially depends on 2’-ribose/deoxyribose identity, but does not depend on 3’-ribose/deoxyribose identity.  Moreover, we have mapped the positions of β’942 in open and closed TL in solution by combining systematic FRET and distance-restrained docking. Top-ranked model solutions placed the representative residue of TL in the secondary channel with a significant difference in position between the open and closed states. Modeled positions change from open state to closed state in a similar manner as suggested by crystal structures. Modeled position of β’942 in open TL is not compatible with the SI3 orientation accommodating closed TL, which suggested that the SI3 module varies in at least two orientational states in response to the conformational cycling of TL in each nucleotide addition step.  Besides, we have developed four procedures to solve specific problems encountered in this work and they may have further applications.  To improve the yield of Cou-labelled β’subunit, a procedure of incorporating Cou into β’ with shortened distance from translation start by using split β’ was developed. This procedure can probably improve the yield of β’ or β (largest and most commonly used RNAP subunits in unnatural amino acid mutagenesis) mutants incorporated with other unnatural amino acids whose incorporation efficiencies have been found low.  To improve the transcriptional activity of Cou-labelled RNAP, a procedure of mutating the amino acid residues adjacent to Cou was developed. This procedure provides an option of introducing an unnatural amino acid into protein interior with minimized loss of protein functions.  To improve the monodispersity of Cou-labelled RNAP, a procedure of one-pot in vivo assembly for Cou-labelled RNAP was developed. This procedure can be easily applied to preparation of RNAP incorporated with other unnatural amino acids, and that can serve as an alternative method to RNAP reconstitution but with a higher protein yield, a higher enzyme activity, and an easier protocol.   To eliminate fluorescence background from non-functional or excess components, an electroelution-based purification procedure for elongation complexes was developed. This procedure can be readily applied to purification of other RNAP-promoter complexes for fluorescence, or non-fluorescence experiments simply by using a fluorescently labelled RNAP complex as marker.