Ubiquitous transcription factors display structural plasticity and diverse functions: NusG proteins - Shifting shapes and paradigms

Abstract

Numerous accessory factors modulate RNA polymerase response to regulatory signals and cellular cues and establish communications with co-transcriptional RNA processing. Transcription regulators are astonishingly diverse, with similar mechanisms arising via convergent evolution. NusG/Spt5 elongation factors comprise the only universally conserved and ancient family of regulators. They bind to the conserved clamp helices domain of RNA polymerase, which also interacts with non-homologous initiation factors in all domains of life, and reach across the DNA channel to form processivity clamps that enable uninterrupted RNA chain synthesis. In addition to this ubiquitous function, NusG homologs exert diverse, and sometimes opposite, effects on gene expression by competing with each other and other regulators for binding to the clamp helices and by recruiting auxiliary factors that facilitate termination, antitermination, splicing, translation, etc. This surprisingly diverse range of activities and the underlying unprecedented structural changes make studies of these "transformer" proteins both challenging and rewarding.

Keywords: NusG; RfaH; antitermination; refolding; transcription; translation.

Figure 1

Structural conservation among NusG family members. A: E. coli NusG (PDB: 2K06, 2JVV) consists of two domains connected by a flexible linker; the NTD (green) has an NGN fold [16], the CTD has a KOW motif [17]. B: Domain organization of NusG homologs in bacteria, archaea, and eukaryotes. The NGNs bind to RNAP and the DNA [18,51,63]; the KOW domains tether transcribing RNAP to other proteins (e.g. Rho and ribosome in bacteria [86,87,107]) the C-terminal repeats (CTR) present in eukaryotes are regulated by phosphorylation and interact with diverse cellular partners [19,108-111]. C: Structural superposition of NGN (left) and KOW (right) domains prepared using Chimera. NGN domain PDB IDs: E. coli (Eco) NusG: 2KO6; E. coli RfaH: 2OUG; P. furiosus (Pfu) Spt5: 3P8B; Human (Hsa) DSIF: 3H7H; KOW PDB IDs: E. coli NusG: 2JVV; E. coli RfaH: 2LCL; P. furiosus Spt5: 3P8B; Human DSIF: 2E70.

Figure 2

RfaH binding site on the transcription complex. A: A TEC cartoon with the template (black) and non-template (blue) DNA strands forming a transcription bubble that encompasses the nucleotides in the active site (near the catalytic Mg²⁺ ion; black sphere). RNAP subunits and RfaH domains are colored as follows: β (light cyan), β′ (light pink), α (grey), ω (light yellow), RfaH NTD (green), and RfaH CTD (cyan). The nascent RNA (red) pairs to the template DNA to form the RNA:DNA hybrid, and exits the complex under the β flap domain; the helix at the tip of the flap domain is shown. The same color scheme is used in other figures. The NTD directly crosslinks to the non-template DNA strand in the TEC and interacts with the β and β′ subunits with the gate loop (GL; deep cyan) and the β′ clamp helices (CH; orange) elements. B: A close-up of a structural model of RfaH/TEC complex [51]. A hydrophobic cavity on NTD interacts with two hydrophobic residues at the tip of the β′ CH (purple; Ile290 and Ile291 in E. coli), the polar and charged residues on the opposite side of the NTD interact with DNA. This model is supported by biochemical analysis of mutationally-altered RNAP and RfaH variants [51,60]. RfaH residues making the key contacts to the β′ CH (Tyr54, Phe56) and DNA (Lys10, Arg16, and Arg73) are shown.

Figure 3

The β′ CH as a universally conserved target site for the initiation and elongation factors. A: Crystal structure of the E.coli RNAP (PDB ID: 4IGC) with the β and β′ (cyan and light pink, respectively), α (grey) and ω (light yellow) subunits shown as ribbons. The active site Mg²⁺ ion (black sphere), the β GL (residues 368 to 376), and the β′ CH (residues 265 to 310) motifs are highlighted. The β′CH serves as a docking site for many initiation and elongation factors; σ forms direct polar interactions largely with the C-terminal part (red) and many indirect interactions covering nearly all of the exposed CH surface in both the E. coli and T. thermophilus RNAP holoenzymes [112,113], whereas RfaH NTD makes van der Waals contacts with the tip of the β′ CH [51]. Archaeal Spt5 proteins make identical contacts to the β′ CH [62,63]. Basal transcription initiation factors Methanocaldococcus jannaschii TFE [57] and Saccharomyces cerevisiae TFIIB [114] also interact with the CH domain, setting up an orchestrated transition from the initiation to the elongation phase through competition for the CH [115]. B: Sequence alignment of the CH domain prepared using DNAStar MegAlign module; identical residues are boxed, regions are color-coded as in panel A. Eco: E. coli; Pfu: P. furiosus; Sce: S. cerevisiae.

Figure 4

RfaH activation by transformation. In the crystal structure of free RfaH (left; 2OUG), the two domains form a large hydrophobic interface that masks the RNAP-binding site on the NTD (green) and captures the CTD cyan) in an α-helical state, in which the key residues interacting with S10 (e.g. Ile146) face the domain interface. By contrast, an isolated CTD folds as a β-barrel (right; 2LCL) in which Ile146 is exposed to bind S10. This dramatic transformation can be induced by artificial separation of the two domains in vitro [86] but requires the recognition of the ops DNA element in the course of transcription. Direct NTD/ops contacts are thought to transduce a signal to the domain interface that weakens the interactions with the CTD, allowing for domain dissociation and subsequent CTD refolding. The freed NTD and CTD can establish the contacts with RNAP and ribosome, respectively, to modulate transcription, translation, and coupling of these processes.

Figure 5

Families of alternative transcription factors. In E. coli, alternative σ factors recognize specific sequences (in double-stranded DNA and on the non-template stand in the transcription bubble) and the β′CH domain to direct RNAP to specific promoters; σ⁷⁰ is the most abundant and mediates transcription of most genes. Following escape from the promoter and release of σ, alternative elongation factors bind to distinct but overlapping elements on the non-template DNA stand and modulate RNA chain elongation and translation; among them, the most abundant NusG mediates transcription of most genes. The well-established competition among the factors from each group controls initiation and elongation phases of transcription. In addition, transition between these phases, the promoter escape and core RNAP recycling after termination, may be modulated by competition between an initiation and an elongation factor for their β′CH/non-template DNA target.

Figure 6

Recruitment strategies of NusG-like proteins. A: A protein in an open conformation can bind to RNAP at any site, although some sequence specificity could be present. B: A protein in a closed, autoinhibited conformation requires a specific target for recruitment. C: A protein which is encoded by the first gene of the controlled operon can be recruited to RNAP as soon as it emerges from the ribosome, if translation and transcription are closely coupled. Examples of RfaH-like regulators which could be recruited in cis and are known to control their resident operons are TAA [104], AnfA1 [103], and Ubx [116].