A nexus for gene expression-molecular mechanisms of Spt5 and NusG in the three domains of life

Abstract

Evolutionary related multisubunit RNA polymerases (RNAPs) transcribe the genomes of all living organisms. Whereas the core subunits of RNAPs are universally conserved in all three domains of life-indicative of a common evolutionary descent-this only applies to one RNAP-associated transcription factor-Spt5, also known as NusG in bacteria. All other factors that aid RNAP during the transcription cycle are specific for the individual domain or only conserved between archaea and eukaryotes. Spt5 and its bacterial homologue NusG regulate gene expression in several ways by (i) modulating transcription processivity and promoter proximal pausing, (ii) coupling transcription and RNA processing or translation, and (iii) recruiting termination factors and thereby silencing laterally transferred DNA and protecting the genome against double-stranded DNA breaks. This review discusses recent discoveries that identify Spt5-like factors as evolutionary conserved nexus for the regulation and coordination of the machineries responsible for information processing in the cell.

Graphical abstract

Fig. 1

Structure of multisubunit RNAPs in the three domains of life. Representative RNAP structures are shown in top views (a, c, and e) and in front views (b, d, and f): bacterial [a and b; Protein Data Bank (PDB) ID 1I6V], archaeal (c and d; PDB ID 2WAQ), and eukaryotic (e and f; PDB ID 1NT9) RNAPs. The universally conserved core subunits are shown in blue, and the subunits specific for archaeal and eukaryotic RNAPs are highlighted in magenta. (g) The universal Tree of Life; the blue circle indicates that ancestral versions of the core RNAP subunits were present in the LUCA of all life, and the magenta circle indicates that the archaeo–eukaryotic subunits were present before the split of the archaeal and eukaryotic domains of life.

Fig. 2

Structure and domain organisation of Spt5-like factors. Organisation of Spt4/5 and NusG (a). Spt5 consists of an NGN domain (highlighted in firebrick red) and one or more KOW (Kyrpidis, Ouzounis, Woese) domains (green), only eukaryotic Spt5 contains two C-terminal repeats (ctr). Eukaryotic and archaeal Spt5 form a complex with Spt4 (wheat); the zinc ion coordinated by Spt4 is illustrated as a sphere. (b) A structural alignment of Spt4/5 NGN from archaea (Methanocaldococcus jannaschii, Mja) and eukaryotes (Homo sapiens, Hsa; S. cerevisiae, Sce) and the NusG NGN domain from bacteria (E. coli, Eco) prepared using VMD (http://www.ks.uiuc.edu/Research/vmd/). The structure of Mja Spt4/5NGN was solved using a dimeric complex of Spt4 (wheat) and Spt5 NGN (firebrick red), and the Sce (mint green) and Hsa (light blue) Spt4/5 NGN structures were solved by crystallising a fusion protein of Spt4 and Spt5. (c) The X-ray structure of Spt4/5 from P. furiosus (Pfu; PDB ID 3P8B) and (d) the X-ray structure of NusG from Aquifex aeolicus (Aae; PDB ID 1NPR) that contains a “mini”-domain (coloured light grey) inserted into the NGN domain at a position similar to Spt4. The mini-domain is not present in all bacterial NusG variants.

Fig. 3

Structure of RNAP–Spt4/5 complexes in the three domains of life. (a and b) Models of RNAP–Spt4/5 complexes in archaea and eukaryotes, respectively; (c) a model of the RNAP–NusG complex in bacteria. The Spt5 NGN domain is located across the DNA binding channel by interacting with the RNAP clamp coiled coil (also known as clamp helices) on one side of the cleft and with the beta gate loop on the opposite side (highlighted with blue broken rectangles). The X-ray structure of a complex encompassing the archaeal RNAP clamp and Spt4/5 NGN from P. furiosus was fitted into the structures of RNAPs (modified from Ref. 34). The Spt5 NGN domain is highlighted in firebrick red; Spt4, in wheat. In the model of the eukaryotic complex, the Spt5 NGN domain does not appear to make contacts with the wall of the DNA binding channel opposite to the clamp because the beta gate loop in the parental RNAPII structure is disordered.

Fig. 4

Spt4/5 locks the template in the DNA binding channel of RNAP in the TEC. Front (a), side (b and e), and top (c) views of the eukaryotic RNAPII–Spt4/5 TEC. This model was built by combining structural information of the archaeal RNAP clamp–Spt4/5 complex with the X-ray structure and single-molecule fluorescence resonance energy transfer data from the yeast RNAPII–DNA–RNA elongation complex (modified from Ref. 34). (d) The Spt4/5 complex (red wedge) locks the DNA–RNA hybrid and the DNA strands forming the transcription bubble into the active site, it thereby denies both dissociation and association of the DNA template (blue line) from the RNAP. As a result, when Spt4/5 associates with the RNAP–DNA–RNA elongation complex, it increases its stability; by contrast, when Spt4/5 associates with free RNAP, it suppresses nonspecific DNA binding. (e) A surface representation of the complex that emphasises how the template is locked into the RNAP [same orientation as in (b)].

Fig. 5

Transcription factor swapping during the transcription cycle. In the archaeal and eukaryotic systems, the binding sites for the initiation factor TFE (TFIIE alpha) and the elongation factor Spt4/5 overlap on the RNAP clamp in mutual exclusive manner. During promoter escape or early elongation, Spt4/5 displaces TFE from the RNAP. In perfect analogy, the bacterial sigma initiation factor can be displaced in vitro by the Spt5 homologue and paralogue NusG and RfaH, respectively. NusG is able to recruit the rho factor, which results in transcription termination. In contrast, RfaH could also displace NusG in vivo, which protects the elongation complex against the recruitment of rho and termination and thereby facilitates the expression of distal genes.

Fig. 6

Spt4/5 facilitates promoter proximal pausing in metazoans. Binding of TFIID to the TATA element of a eukaryotic class II promoter triggers a recruitment cascade that results in the initiation of transcription. However, the association of Spt4/5 and NELF with RNAPII in the early elongation complex leads to promoter proximally stalled elongation complexes. Activation of kinases including P-TEFb and Bur-1/Bur-2 leads to the phosphorylation of the RNAPII C-terminal domain, Spt4/5, and NELF, which releases the stalled complexes and induces robust gene expression. Many class II genes in metazoans harbour promoter proximally stalled initiation complexes, and it is possible that this mechanism operates on a global level. Phosphorylation events are highlighted with an orange “P”.

Fig. 7

Transcription and translation are coupled via NusG in prokaryotes. (a) Structural model of the RNAP–Spt4/5–S10 elongation complex. The S10 protein (light blue) of the 30S ribosomal subunit forms a complex with the KOW domain (green) of Spt5, and thus, the ribosome is ideally positioned to interact with the mRNA template (cyan). This model was built by combining the X-ray structure of the NusG KOW–S10 complex from E. coli (PDB ID 2KVQ), the structure of the archaeal Spt4/5 complex from P. furiosus (PDB ID 3P8B), and a model of the RNAPII–Spt4/5 NGN complex from S. cerevisiae. The components are colour coded according to the key in the figure. The RNA interacts with the RNAP stalk (Rpo4/7 or RPB4/7) during transcription elongation, but the RNA species included in the X-ray structure (PDB ID 1NT9) was too short to observe this interaction. (b) Mechanisms of coupled transcription–translation. The NusG KOW domain interacts with the rho factor and ribosomal protein S10 in a mutual exclusive manner. RNAP–NusG elongation complexes are able to recruit rho, which can lead to (pre-) mature termination at rho termination sites. Ribosomal protein S10 can bind to the NusG KOW domain and thereby prevent the recruitment of rho. Thus, the efficient coupling of transcription and translation (i.e., of RNAP and ribosome) prevents premature termination. Following translation termination, the ribosome dissociates from the transcript and makes the NusG KOW domain accessible for rho binding, which promptly leads to transcription termination. Likewise, the NusG paralogue RfaH can recruit ribosomes, protect against rho, and facilitate the expression of distal genes.