Multiple personalities of the RNA polymerase active centre

Abstract

Transcription in all living organisms is accomplished by highly conserved multi-subunit RNA polymerases (RNAPs). Our understanding of the functioning of the active centre of RNAPs has transformed recently with the finding that a conserved flexible domain near the active centre, the trigger loop (TL), participates directly in the catalysis of RNA synthesis and serves as a major determinant for fidelity of transcription. It also appears that the TL is involved in the unique ability of RNAPs to exchange catalytic activities of the active centre. In this phenomenon the TL is replaced by a transcription factor which changes the amino acid content and, as a result, the catalytic properties of the active centre. The existence of a number of transcription factors that act through substitution of the TL suggests that the RNAP has several different active centres to choose from in response to external or internal signals.A video of this Prize Lecture, presented at the Society for General Microbiology Annual Conference 2014, can be viewed via this link: https: //www.youtube.com/watch?v=79Z7iXVEPo4 TRIGGER LOOP: A NEW CATALYTIC DOMAIN OF THE RNA POLYMERASE ACTIVE CENTRE Multi-subunit RNAPs are the enzymes that perform transcription in all living organisms. RNAPs are highly conserved, and emerged before the divergence of the bacteria and archaea/eukaryote lineages. All RNAPs share the invariantly conserved catalytic core of five subunits: b, b9, 2a and v (bacterial nomenclature is used throughout). The two largest subunits, b and b9, form the catalytic cleft where the reaction of addition of nucleoside monophosphates (NMPs) to the growing RNA takes place. As with many other nucleic-acid-managing enzymes (Steitz & Steitz, 1993) and all nucleic-acid-polymerizing enzymes (Steitz, 1998), RNAP uses a two-metal-ion (Mg 2+ ) mechanism to catalyse the phosphotransfer reaction. One of the metal ions, Mg 2+ I, is chelated by the invariant triad of aspartates of the b9 subunit, whilst the other one, Mg 2+ II, is brought by substrates, e.g. by the incoming nucleoside triphosphate (NTP). Mg 2+ I stabilizes the negative charge on the attacking oxygen of the hydroxyl group of the 39 NMP of RNA. Mg 2+ II assists the leaving of the pyrophosphate, and both metal ions ligate the non-bridging oxygen to stabilize the pentacovalent transition state. Substitutions in the aspartate triad lead to almost full inactivation of the enzyme (Zaychikov et al., 1996).The emergence of the first crystal structure of multi-subunit RNAP (Zhang et al., 1999) ignited structure-based functional analysis of the enzyme by many researchers. We were interested in the disordered loop (referred to initially as the G-loop and, later, as the TL) of the highly conserved G domain of the b9 subunit. In the structure, this disordered region was located .20 Å from Mg 2+ I. However, we found that deletion of this loop had a dramatic effect on catalysis by slowing down NMP addition~10 4 times -an effect close to that of mutations in the aspartate triad. Three years later we foun...

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

“…Altogether, these observations extend the concept of factor-dependent exchange of RNAP activities in response to various regulatory stimuli (16,21,34), and suggest that various secondary channel regulators may play a general role in metal homeostasis and, more broadly, in adaptation to complex ecological niches.…”

“…The TL in the RNAP active site has previously been implicated in functional interplay with Gre factors, which should replace it to stimulate the RNA cleavage reaction (16,29,34). To reveal whether the TL is important for pause stimulation by Gfh factors, we analyzed mutant Dra RNAP with a deletion in the TL (Fig.…”

Dynamics of GreB-RNA polymerase interaction allow a proofreading accessory protein to patrol for transcription complexes needing rescue

“…Nevertheless, competition experiments between GreB and DksA show that each protein's activity is unimpeded in the excess (10-to 300-fold) presence of the other protein, raising the possibility that the proteins are recruited to distinct subpopulations of ECs (18,20). In contrast, other observations have been taken to suggest that multiple SC factors can bind simultaneously to RNAP; such simultaneous binding is inconsistent with simple binding competition (19,20,22).…”

“…In particular, a combination of crystallographic data and structural modeling suggests that when GreB binds in the SC, it is likely to insert into the RNAP active site in a manner that inhibits nucleotide addition to the nascent transcript (13,(17)(18)(19)(20). However, functional studies have not demonstrated inhibition by wild-type Gre proteins in vitro (17,20), raising the question of how the proteins can stimulate transcript cleavage but not interfere with transcript elongation.…”

“…elongation and termination (18)(19)(20). Nevertheless, competition experiments between GreB and DksA show that each protein's activity is unimpeded in the excess (10-to 300-fold) presence of the other protein, raising the possibility that the proteins are recruited to distinct subpopulations of ECs (18,20).…”