A Nexus for Gene Expression—Molecular Mechanisms of Spt5 and NusG in the Three Domains of Life

1Life is strictly dependent on the faithful replication of the genetic information and its expression through the synthesis of RNA and protein. This is reflected in the evolutionary conservation of the machineries that carry out these functions and in their regulation. Replication, transcription, and translation are not independent of another but are coordinated in a dynamic fashion that adjusts the needs of one with the yield of the other, and overall enables maximum energy efficiency and protection of genome integrity.

2Transcription of all cellular genomes is carried out by multisubunit RNA polymerases (RNAPs), all of which are derived from one ancestral enzyme present in the last universal common ancestor (LUCA) (Fig. 1). This evolutionary relationship is reflected in the homology of RNAP subunit sequence and structure, overall RNAP ternary architecture, and molecular mechanisms by which RNAPs transcribe RNA in a DNA-template-dependent manner (Fig. 1 and Table 1). Interestingly, the same conservation does not extend to the basal transcription factors that assist RNAP during the transcription cycle. For example, whereas all RNAPs face the similar mechanistic challenges during transcription initiation (such as RNAP recruitment, DNA strand separation, initiation of RNA polymerisation, and promoter escape), the bacterial and archaeal/eukaryotic RNAPs utilise nonhomologous factors to facilitate these processes (Table 1). The bacterial RNAP utilises sigma factors, whereas eukaryotic RNAPII uses the basal factors TBP (TATA binding protein) or TFIID [TBP plus TAFs (TBP-associated factors)], TFIIA (transcription factor IIA), TFIIB, TFIIE, TFIIF, and TFIIH. The archaeal RNAP is a streamlined version of the RNAPII system and utilises TBP, TFB (homologous to TFIIB), and TFE (homologous to TFIIE alpha).

3The fact that bacteria and archaea/eukaryotes make use of nonhomologous factors for initiation strongly suggests that the ancestral RNAP of LUCA did not utilise any of these factors but rather initiated transcription either factor-independently, or aided by a factor that subsequently was lost in evolution. There is a precedence for the former mechanism since (evolutionary unrelated) single subunit RNAPs (e.g., bacteriophage T7 RNAP) can initiate transcription by directly recognising the promoter DNA in a sequence-specific manner.

4During elongation, multisubunit RNAPs frequently pause and move in a retrograde direction along the template by backtracking, which can regulate transcription and contribute to its fidelity, but also be detrimental to productive transcription. Backtracked complexes are rescued by transcript cleavage factors that associate with RNAP and retune its active site, which results in a cleavage of the transcript and a new RNA 3′ terminus competent for catalysis and elongation. Similar to the initiation phase of transcription, the challenges that RNAPs need to overcome during elongation are identical, but the transcript cleavage factors that solve the problem are not homologous. In bacteria, Gre factors stimulate transcript cleavage, and in archaea/eukaryotes, TFIIS/TFS fulfil this function. Gre and TFIIS/TFS are not homologous and adopt different structures; however, they interact with RNAP in the same manner by inserting two juxtaposed acidic residues into the active centre through the NTP entry pore. The lack of bona fide homology suggests—despite the compelling similarity of the mechanism—that the LUCA RNAP did not utilise any of the factors.

5During termination of transcription, the stable elongation complex has to dissociate in order to release the transcript and template and make RNAP available for the next round of transcription. The elongation complex has to undergo substantial conformational changes during termination (such as an opening of the RNAP clamp) that are not necessarily energetically favourable. In bacteria, this energy is provided by RNA secondary structure formation by Watson–Crick base pairing of intrinsic terminators or by the rho helicase by virtue of ATP hydrolysis. Termination by eukaryotic RNAPII is more complex and involves both polyadenylation of the transcript and an exonuclease (Xrn2/Rat1) that, despite being unrelated to rho, translocates along the RNA towards the RNAP and terminates transcription reminiscent of rho. Eukaryotic RNAPIII and archaeal RNAP are able to terminate transcription independently of RNA secondary structures or factors. Thus, similar to the transcription initiation and elongation cleavage factors, none of the termination factors are evolutionary conserved across the three domains of life.

6In summary, it appears that a highly conserved RNAP system is almost exclusively aided by evolutionary nonrelated transcription factors. One important exception to this observation is Spt5/NusG, and the key functions that this factor have in gene expression might explain its universal evolutionary conservation.

7Eukaryotic and archaeal Spt5 and their bacterial homologue NusG display an extensive sequence and structural homology and associate with their cognate RNAPs via evolutionary conserved binding sites. The organisation of Spt5-like proteins is modular, and all factors encompass an NGN (NusG N-terminal) domain and one (bacteria and archaea) or multiple (eukaryotes) KOW domain(s) (Fig. 2a). The NGN domain interacts with RNAP and stimulates its processivity, whereas the KOW domains serve as a recruitment platform for accessory factors (Table 2). NusG from bacteria is a monomeric transcription factor. Spt5 from eukaryotes and archaea forms a heterodimeric complex with Spt4, a small protein that stabilises the Spt5 NGN domain structure but, otherwise, has an unknown function. The evolutionary conservation of Spt5 and NusG is apparent from their amino acid sequence but becomes even more prominent when comparing the structures of the NGN domains (Fig. 2). Mutational analysis of the Spt5 and NusG NGN domains have identified a conserved hydrophobic depression that functionally interacts with the RNAP clamp coiled coil (“CC”, also referred to as clamp helices “CH” in bacteria) on one side of the DNA binding channel. The X-ray structure of a complex between a recombinant fragment of the Pyrococcus furiosus RNAP clamp with Spt4/5 confirms this binding site. As shown for the bacterial NusG paralogue RfaH, NGN domains also make contact with the RNAP beta gate loop (“betaGL”), which is located opposite to the clamp CC across the DNA binding channel (Fig. 3). Via these two interactions, the NGN domains of Spt5-like factors (i) lock the DNA–RNA hybrid into the active site and (ii) separate the upstream and downstream portions of the template (Fig. 4). A cryo-electron microscopy structure of the entire archaeal RNAP–Spt4/5 complex confirms that the NGN domain of Spt5 and, by inference, its homologues NusG and RfaH span across the DNA binding channel of RNAP.

8Spt5-like factors bind to the RNAP clamp, a structurally well conserved flexible module that closes over the DNA binding channel by a rotation of up to 30° relative to the RNAP core. The position and possibly movement of the clamp can be altered by the archaeal/eukaryote-specific RNAP stalk (consisting of the Rpo4/7 and RPB4/7 subunits; Fig. 1c–f), which has been hypothesised to play a role during the loading of the template into the active site (open complex formation) and which is important to maintain high transcription processivity. The translocation/elongation mechanism of RNAPs is facilitated by the bridge helix and trigger loop in the active site. Since both elements are connected to the inside of the clamp, even a slight reposition of the latter can have substantial impact on the elongation properties of the transcription elongation complex (TEC); that is, Spt5-like factors could alter the catalytic properties of RNAP via an allosteric mechanism.

9By binding across the DNA binding channel of RNAP, Spt4/5 (and NusG and RfaH) has the potential to affect RNAP in two opposite ways (Fig. 4). When Spt4/5 associates with elongating RNAPs, it could deny the dissociation of RNAP-bound DNA and could thereby increase the stability of the elongation complex and stimulate transcription processivity. When Spt4/5 associates with free RNAP, it could deny association of promoter DNA with the DNA binding channel of RNAP, prevent RNAP recruitment, and thereby repress transcription (Fig. 4). The stimulatory property for transcription processivity of RNAP by NusG in bacteria and Spt4/5 in archaea is well documented, and a recent article also supports the second, inhibitory, mechanism. Preincubation of Spt4/5 with archaeal RNAP efficiently inhibits RNAP recruitment and transcription initiation in a minimal in vitro transcription system.

10The archaeal RNAP requires only two basal transcription factors for promoter-directed transcription, TBP and TFB. TBP recognises the TATA element of the promoter and bends the DNA by approximately 90°; this DNA–TBP complex is recognised by TFB, which in turn recruits RNAP and transcription initiation commences. In contrast, the Spt4/5–RNAP complex cannot be recruited to the DNA–TFB–TFB complex, and transcription initiation is repressed.

11The answer to this question is the transcription initiation factor TFE. TFE is homologous to TFIIE alpha in the eukaryotic RNAPII system and of C82 in RNAPI. TFE associates with RNAP and stabilises the open transcription initiation complex by two mechanisms involving an allosteric effect on the RNAP clamp and by interacting with the nontemplate strand of the promoter. In addition, TFE can be cross-linked downstream of promoter sequences, which suggests that it can be retained on RNAP elongation complexes after promoter escape. However, the binding site of TFE or the molecular mechanisms underlying its activities have remained obscure until recently. Grohmann et al. used a Förster resonance (fluorescence resonance energy transfer) system to identify the TFE binding site on RNAP in the complete archaeal transcription initiation complex. Both the TFE winged helix domain and the Spt5 NGN domain interact with the RNAP clamp coiled coil. The two binding sites are overlapping, and correspondingly, Spt4/5 and TFE compete for RNAP binding. Importantly, the relative binding affinities of the factors are context dependent: TFE prevails over Spt4/5 in the initiation complex, whereas Spt4/5 prevails over TFE in the elongation complex. As a result, TFE can efficiently prevent the inhibitory effect of Spt4/5 on transcription initiation.

12All RNAPs bind DNA in a nonsequence-specific manner, which can critically quench or reduce the concentration of “free” RNAP available for promoter-directed transcription. Association of RNAP with Spt4/5 has the potential to reduce this effect, and since the repression by Spt4/5 is negated by TFE at the promoter, the overall outcome is an increase in promoter specificity of RNAP. In addition, or alternatively, the transition between the initiation and elongation phases of transcription—referred to as promoter escape—may be aided by the factor swap. One of the intrinsic conflicts during transcription initiation is the apparent need for high-affinity interactions between RNAP and promoter DNA to ensure efficient RNAP recruitment and the ability to readily release the promoter-bound RNAP necessary to enable a high firing rate of the promoter. This is made possible by the binding mode of initiation factors to RNAP where multiple contacts are made with a combined high affinity. Conformational changes in the initiation complex during promoter escape disrupt the individual interactions in a stepwise manner. The distinct conformational states of RNAP in the initiation and elongation complexes may be stabilised by associated transcription factors, and the energy barriers for the changes between the conformational states can be lowered by association with transcription factors. Thus, Spt4/5 displacing TFE could stimulate promoter escape (Fig. 5). Alternatively, Spt4/5 could ensure that RNAP-bound TFE during early elongation is efficiently displaced and replaced by Spt4/5, resulting in a high-processivity RNAP–Spt4/5 TEC. Whole genome occupancy profiling of RNAPII and transcription initiation and elongation factors in Saccharomyces cerevisiae has revealed a relatively sharp transition between initiation and elongation factors (including Spt4/5) at approximately 150 bp downstream of the transcription start site. Whereas this position is too far downstream to qualify as promoter escape, it supports the notion of an efficient swap between initiation and elongation factors. Interestingly, the bacterial transcription initiation factor sigma, despite not being evolutionary related to TFE, also interacts with the RNAP CH. Sigma can, like TFE, remain associated with RNAP during early elongation and induce pausing. Importantly, NusG and RfaH, like Spt5, interact with the RNAP clamp CH, and both can compete with sigma for RNAP binding (Fig. 5).

13In summary, the mechanism of competition binding between initiation and elongation factors for RNAP is universally conserved in evolution.

14In the bacterial system, NusG is an essential general transcription factor that has pleiotropic effects on transcription elongation. NusG is a component of the antitermination complex that converts RNAP into a termination-resistant form, which is able to read through early intrinsic termination signals in, for example, ribosomal RNA (rrn) operons and during late gene expression of the bacteriophage lambda. NusG stimulates processivity by suppressing transcription pausing (also referred to as antipausing), but it also acts in concert with rho to facilitate termination. rho itself interacts with RNAP and is recruited to the TEC via physical interactions with the NusG KOW domain (Figs. 5 and 7). Thus, NusG association with RNAP enhances elongation but also directly contributes to termination. The whole genome occupancy profile of NusG demonstrates that it is bound to TECs on nearly all genes, protein encoding and noncoding transcription units. NusG appears to be recruited to genes in a stochastic fashion downstream of the promoter, and it is overrepresented on longer operons. Whether the latter is due to the fact that TEC on long genes have more opportunities to recruit NusG or whether NusG association with RNAP enables the TEC to transcribe longer genes is unclear. RfaH is a nonessential sequence-specific paralogue of NusG that induces the expression of genes downstream of the operon polarity suppressor (ops) DNA element. Transcription of the ops sequence triggers recruitment of RfaH to TEC and directs the transcription of a small subset of operons involved in bacterial virulence. On the sequence level, RfaH and NusG are closely related, and this homology extends to the structural level with respect to their NGN domains. However, whereas the NusG C-terminus is a typical KOW domain entirely consisting of beta sheets, the RfaH C-terminal domain in full-length RfaH is a radically different all-alpha helical fold. RfaH, despite being present in the cell at much lower concentration than NusG, excludes NusG from binding RNAP once the TEC has transcribed through the ops element. Whole genome occupancy profiles demonstrate that RNAP and NusG peaks overlap just downstream of the promoter on the rfb operon—indicative of NusG recruitment immediately after transcription initiation where the swap of sigma and NusG has occurred. A second swap is executed with a sharp transition at approximately 1 kb when NusG is replaced by RfaH. This reduces transcription termination facilitated by rho by at least two independent mechanisms, a stimulation of transcription processivity by converting RNAP into a high-processivity (pause-resistant state) TEC similar to NusG and by preventing the recruitment of rho via the NusG KOW domain. The latter is demonstrated by the occupancy profile of rho on the rfb operon. The net outcome of the second swap (between NusG and RfaH) in the very early stages of transcription elongation is the efficient transcription of genes distal to the ops sequence.

15In summary, both NusG and RfaH interact with the RNAP using a conserved mechanism, but the two factors have opposing regulatory roles. NusG in concert with rho represses the expression of “foreign” genes (see below), while RfaH inhibits rho action on RNAP and thereby enhances the expression of “foreign” genes such as the rfb operon.

16In eukaryotes, Spt4/5 is associated with RNAPII on most, if not all, genes; Spt5 is an essential gene in S. cerevisiae, whereas Spt4 is dispensable at permissive temperatures. This is in good agreement with results from the archaeal system, which demonstrate that Spt4 has a stabilising effect on the Spt5 NGN domain but is not strictly required for its binding to RNAP nor its stimulatory effect on processivity. Eukaryotic Spt5 variants contain four to six copies of KOW domains (in comparison to one in archaea and bacteria) and two C-terminal repeat regions that are subject to phosphorylation but not required for cell viability in yeast. The additional KOW domains serve as binding or recruitment sites for a plethora of factors including NELF (negative elongation factor) and kinases such as P-TEFb (positive transcription elongation factor b) and Bur-1/Bur-2, factors that are involved in chromatin remodelling such as Spt6 and FACT, and RNA processing factors such as the mRNA capping enzyme and the cap methyl transferase (Table 2).

17The classical paradigm of transcription control—regulation is chiefly controlled by the recruitment of the RNAPII to the promoter—was established using the yeast transcription machinery. Early observations of the regulation of the hsp70 heat shock gene in a metazoan system, Drosophila melanogaster, suggested that its transcription was not regulated at the level of RNAPII recruitment. Rather, TECs were paused (also referred to as stalled or poised) proximal to the promoter approximately 40 bp downstream of the transcription start site. The complexes were catalytically competent but not able to penetrate into the downstream gene. Signalling events induced by heat shock lead to the activation of the kinase P-TEFb that phosphorylates multiple components of the elongation complex including Spt4/5, NELF, and RNAPII (Fig. 6). This results in the release of the poised elongation complexes and robust expression of the heat shock genes. The regulatory advantage of these mechanisms is a fast induction achieved by uncoupling of RNAP recruitment and transcription. In the last 5 years, it has become apparent that many, if not most, genes in metazoans harbour promoter proximally stalled RNAPII and are therefore likely to be subject to this regulation. Promoter proximal stalling could have other functions than regulation such as mRNA quality control and coupling between transcription and mRNA processing. A delay of elongation complexes could thus allow the recruitment of RNA modification enzymes such as the capping enzyme, and elongation would commence only if the capping is completed. Both Spt4/5 and NELF are required for promoter proximal pausing, but the molecular mechanisms are still not understood. Spt4/5 is a key candidate for directly inhibiting RNAP activity because its binding to RNAP alters the DNA-binding properties of the enzyme and possibly induces conformational alterations in the active centre (see above) that, in theory, could pause transcription elongation. The significance of the position of the paused complexes at + 40 is unclear, even though it seems compelling that human RNAPII via the RPB4/7 complex is interacting with approximately 40 nucleotides of the transcript and that the recruitment of eukaryotic Spt4/5 to RNAPII is enhanced, if not dependent on, interactions between the Spt5 KOW domains and the RNA transcript.

18In summary, the Spt4/5 complex is associated with many, if not all, class II genes in eukaryotes, and it plays a pivotal role for a eukaryote-specific mechanism that regulates the processivity of transcription—promoter proximal stalling. In addition, like its prokaryotic counterpart, it is involved in the recruitment of accessory factors that regulate gene expression, alter the availability of the chromatin template, and are involved in the processing of the nascent transcript. The function of Spt4/5 in eukaryotes is not restricted to class II genes since Spt4/5 also directly associates with RNAPI and modulates its processivity both positively and negatively.

19In both prokaryotic domains, bacteria and archaea, transcription and translation are coupled. In bacteria, the rates of transcription and translation are correlated over a broad range of growth rates, which keeps the ratio between the two elongation rates at 3 (three nucleotides for every one amino acid). As a result, the transcription yield is correlating with the translation needs on different genes and during varying growth rates. In effect, the elongation rate of transcription is influenced by the presence of ribosomes and their elongation rate of translation; the average elongation rate of RNAPs can be increased by cotranslating ribosomes, possibly because the latter reduce pausing and backtracking of RNAP. Ribosomes whose elongation rates have been decreased by antibiotic action in turn cause a decrease in transcription elongation rates of RNAP by virtue of their intrinsic propensity to pause and backtrack. In both situations, little mRNA is exposed and available for recognition by rho; that is, the gene is protected against rho-dependent termination. These phenomena could be explained by the motions of two molecular motors, one of which uses the product of the other as template (mRNA). Recently, it has emerged that the NusG KOW domain physically interacts with ribosomal protein S10 (first characterised as antitermination factor NusE) and that this interaction could tether elongating ribosomes and RNAPs (Fig. 7a). In principle, this interaction could also enhance ribosome recruitment during translation initiation. Since S10 competes with rho for the binding to the NusG KOW domain, the coupling of transcription and translation protects the TEC against rho-dependent termination (Fig. 7). Translation is terminated at the 3′ end of operons, and S10 dissociates from RNAP-bound NusG, which makes the KOW domain available for the recruitment of rho and subsequent efficient transcription termination. In summary, Spt5-like factors have the potential to coordinate transcription and translation in prokaryotes, bacteria and archaea.

20Bacterial genomes contain a considerable load of horizontally transferred or “foreign” DNA including prophages, insertion sequence elements, and K-island clusters. Many of these elements can be deleted to obtain reduced genome strains, which sometimes have beneficial biotechnological properties and different requirements for essential genes than their parental strains. In the synthetic Escherichia coli strain MDS42, 14% of the genome of the parental strain MG1655 has been deleted. In contrast to MG1655, NusG (and NusA) is not essential for cell viability in MDS42. Moreover, deletion of the lambdoid prophage rac from MG1655 is sufficient to make NusG dispensable. Considering that MDS42 has a dramatically increased (∼ 104×) resistance to the highly selective inhibitor of the rho termination factor (bicyclomycin), it is possible that the function of NusG that makes it indispensable in the wild-type strain is due to its role as a rho cofactor for rho. In other words, NusG in conjunction with rho silences rac gene expression, which otherwise would kill the cell. A whole genome expression analysis reveals that bicyclomycin treatment predominantly upregulates the expression of foreign DNA, which suggest that rho represses its expression. Since rho to a large extent relies on NusG as cofactor, NusG may play an important role for the silencing of foreign DNA on a global level.

21Transcription and replication occur simultaneously, and collisions between the TEC and the replication fork not only are inevitable but also occur on a regular basis. Since the replisome moves at a greater speed (20×) than the TEC, even codirectional collisions happen frequently, and these can lead to double-stranded DNA breaks (DSBs). Two recent articles show that rho-dependent termination protects against DSBs by removing arrested elongation complexes. In particular, the DSBs caused by codirectional collisions were dependent on backtracked TECs. The cell has evolved a range of strategies to prevent DSBs caused by backtracked TEC. Transcription factors that improve transcription processivity and suppress backtracking and factors that reactivate backtracked TEC by transcript cleavage reduce the occurrence of DSB. Efficient coupling of transcription and translation, that is, of RNAP and ribosomes, suppresses backtracking and reduces DSB. Transcription termination factors such as rho and its cofactors that remove backtracked and arrested TEC from the genome also protect against DSB. The common denominator of all these strategies is NusG: it increases processivity, efficiently couples transcription and translation, and promotes termination in conjunction with rho.

22Spt5-like transcription factors are universally conserved in evolution and serve multiple functions in the three domains of life.

23The most basic function of Spt5-like proteins that is common to all transcription systems is the stimulation of transcription processivity carried out by the universally conserved Spt5 NGN domain. The exact composition of early genomes is unclear; they could have consisted of either DNA or RNA, either in single-stranded or in double-stranded form. The catalytic centre of multisubunit RNAPs can utilise single-stranded DNA as template and, under special circumstances, RNA as template for transcription. For example, RNAPII can transcribe the hepatitis virus delta RNA genome, and bacterial RNAP uses the noncoding 6S RNA regulator as template for RNA-dependent RNA polymerisation. However, the processivity of transcription on these templates is severely impaired, possibly because the interactions between RNAP and single-stranded DNA or RNA are weaker as compared to double-stranded DNA. Likewise, ancestral versions of multisubunit RNAPs were likely capable of using nonduplex DNA as template. The ancestral version of Spt5 might have been crucial for robust RNAP function using these early “poor” genome templates, and thus, Spt5 could have provided a crucial selective advantage for its host. Experimental evidence supports this notion, since archaeal Spt4/5 strongly enhances the processivity of its cognate RNAP using single-stranded template DNA strand. Not unlike the RNAP stalk, the Spt5 ancestor may have played a crucial role for the expression of long genes and, during evolution, even have “permitted” an increase in gene or operon length and thereby assisted an increase in the complexity of the genetic repertoire of the organism. Once established as an integral component of TECs, other functions including the recruitment of transcription termination factors (such as rho in bacteria), the coupling of RNAPs to ribosomes (in prokaryotes), and nascent RNA processing (in eukaryotes) might have emerged over time due to the advantageous location of Spt5 in the TEC: across the DNA binding channel of RNAP, proximal to the nascent transcript, and bound to RNAP in a reversible manner. All recruitment sites for exogenous factors are located in the Spt5 KOW domain(s). The expansion of the number of KOW domains in evolution (e.g., one in prokaryotes, four in yeast, and six in humans) seems in-line with the notion that they facilitate more elaborate, later additions to the functionality of Spt5-like factors.