pH-dependent conformational switch activates the inhibitor of transcription elongation

Laptenko, Oleg; Kim, Seung-Sup; Lee, Jookyung; Starodubtseva, Marina; Cava, Felipe; Berenguer, José; Kong, Xiang‐Peng; Borukhov, Sergei

doi:10.1038/sj.emboj.7601094

Cited by 61 publications

(76 citation statements)

References 33 publications

(57 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It was shown to have a relatively low inhibitory effect on TLdependent activities (Laptenko et al, 2006). However, by analogy with Gre, the switch between TL and Gfh1 active centres may happen in a controlled manner in response to some signals which have not yet been found.…”

Section: Multiple Active Centres Of Rnapmentioning

confidence: 99%

Multiple personalities of the RNA polymerase active centre

Zenkin

2014

Microbiology

View full text Add to dashboard Cite

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...

show abstract

Section: Multiple Active Centres Of Rnapmentioning

confidence: 99%

Multiple personalities of the RNA polymerase active centre

Zenkin

2014

Microbiology

View full text Add to dashboard Cite

show abstract

“…Similar to GreA, Gfh1 was proposed to bind metal ions in the RNAP active site by acidic residues from the tip of its N-terminal domain (NTD) (Fig. 1B), but to stabilize them in catalytically nonproductive positions (20,21). In addition, it induces major changes in RNAP conformation (RNAP "ratcheting"), including opening of the clamp/shelf module and kinking of the BH (9,22).…”

mentioning

confidence: 99%

“…In addition, it induces major changes in RNAP conformation (RNAP "ratcheting"), including opening of the clamp/shelf module and kinking of the BH (9, 22). The activity of Gfh1 is regulated by a pH-dependent rotation of its C-terminal domain, resulting in its activation at low pH values (20).Deinococcus radiodurans (Dra) is a highly radioresistant and stress-resistant mesophilic bacterium closely related to Tth. It encodes two Gfh factors, Gfh1 and Gfh2, whose cellular functions remain unknown.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

“…S1). The Gfh1 protein from Thermus thermophilus (Tth) was shown to inhibit all RNAP activities, including nucleotide addition and RNA cleavage (18)(19)(20)(21). Similar to GreA, Gfh1 was proposed to bind metal ions in the RNAP active site by acidic residues from the tip of its N-terminal domain (NTD) (Fig.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

Esyunina

Agapov

Kulbachinskiy

2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Transcriptional pausing has emerged as an essential mechanism of genetic regulation in both bacteria and eukaryotes, where it serves to coordinate transcription with other cellular processes and to activate or halt gene expression rapidly in response to external stimuli. Deinococcus radiodurans, a highly radioresistant and stressresistant bacterium, encodes three members of the Gre family of transcription factors: GreA and two Gre factor homologs, Gfh1 and Gfh2. Whereas GreA is a universal bacterial factor that stimulates RNA cleavage by RNA polymerase (RNAP), the functions of lineage-specific Gfh proteins remain unknown. Here, we demonstrate that these proteins, which bind within the RNAP secondary channel, strongly enhance site-specific transcriptional pausing and intrinsic termination. Uniquely, the pause-stimulatory activity of Gfh proteins depends on the nature of divalent ions (Mg 2+ or Mn 2+ ) present in the reaction and is also modulated by the nascent RNA structure and the trigger loop in the RNAP active site. Our data reveal remarkable plasticity of the RNAP active site in response to various regulatory stimuli and highlight functional diversity of transcription factors that bind inside the secondary channel of RNAP.RNA polymerase | transcriptional pausing | Gfh factors | Deinococcus radiodurans | stress response C ellular RNA polymerases (RNAPs) are complex molecular machines whose activity during transcription is regulated by DNA-and RNA-encoded signals, protein factors, small molecules, and inhibitors. The catalytic cycle of RNAP can be interrupted by pauses of various natures that play important roles in genetic regulation in all organisms, from the classic systems of transcription attenuation and their variations in bacteria (1) to recently discovered widespread promoter-proximal pausing in eukaryotes (2). The pausing serves to activate or repress transcription rapidly at specific genomic sites in response to regulatory stimuli and to coordinate RNA synthesis with other genetic processes (e.g., DNA replication and repair, RNA translation in bacteria) (1,(3)(4)(5)(6)(7)(8).Recent structural and biochemical studies revealed distinct RNAP conformations corresponding to different functional states of the transcription elongation complex (TEC) during nucleotide addition, RNA proofreading, and pausing (9-11). The control of structural transitions between these states likely underlies the function of multiple regulatory factors acting on RNAP. However, the roles of individual RNAP conformations in transcription and the mechanisms of their switching by transcription factors remain only partially understood. During transcription, RNAP holds the DNA template and the RNA transcript within its main channel, whose opening is controlled by the mobile clamp/shelf module and the flap domain of RNAP (9-11). Nucleotide addition and TEC translocation depend on alternating cycles of the folding of the trigger loop (TL) and kinking of the bridge helix (BH) in the RNAP active site (9-11) (Fig. 1A). Analysis of the stru...

show abstract

Gfh factors and NusA cooperate to stimulate transcriptional pausing and termination

et al. 2017

View full text Add to dashboard Cite

Lineage-specific Gfh factors from the radioresistant bacterium Deinococcus radiodurans, which bind within the secondary channel of RNA polymerase, stimulate transcriptional pausing at a wide range of pause signals (elemental, hairpin-dependent, post-translocated, backtracking-dependent, and consensus pauses) and increase intrinsic termination. Universal bacterial factor NusA, which binds near the RNA exit channel, enhances the effects of Gfh factors on termination and hairpin-dependent pausing but do not act on other pause sites. It is proposed that NusA and Gfh target different steps in the pausing pathway and may act together to regulate transcription under stress conditions. Thus, transcription factors that interact with nascent RNA in the RNA exit channel can communicate with secondary channel regulators to modulate RNA polymerase activities.

show abstract

pH-dependent conformational switch activates the inhibitor of transcription elongation

Cited by 61 publications

References 33 publications

Multiple personalities of the RNA polymerase active centre

Multiple personalities of the RNA polymerase active centre

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

Gfh factors and NusA cooperate to stimulate transcriptional pausing and termination

Contact Info

Product

Resources

About