Chapter 8 : Processive Antitermination

Genomic context of PA systems. This figure schematically illustrates the transcripts regulated by the λN, put, rRNA, and EAR RNA-based antitermination systems. (A) Phage λ early transcripts are initiated from two divergently facing promoters with boxA/B nut elements found early in the transcripts. The λN protein is encoded by the first gene in the left early transcript. RNAP complexes associated with λN bypass multiple terminators in both transcripts. Using a different mechanism, the λQ protein promotes antitermination of the late transcript by binding to DNA near the late promoter and promoting a terminator-resistant configuration of RNAP. (B) Phage HK022 early transcripts are similar to phage λ, although they include put elements early in each transcript, which trigger λN-independent antitermination. Additional Rho-dependent terminators are likely present in these transcripts, although they have not been specifically characterized and are therefore not indicated here. (C) A representative E. coli rRNA operon is shown, containing boxA/B/C elements immediately downstream of the P2 promoter. These elements promote read-through of Rho-dependent termination in the noncoding rRNA genes. (D) Several intrinsic terminators have been demonstrated in the B. subtilis eps operon, which codes for biosynthesis of biofilm exopolysaccharides. The eps-associated RNA (EAR) is found within the epsBC intergenic region and promotes read-through of the terminators within the operon. Intrinsic terminators are shown as sticks with empty circles, and Rho termination regions are shown as sticks with wavy lines, both in red. RNA elements involved in antitermination are shown in blue, and proteins and protein-coding genes involved in antitermination are shown in green. Elements are not shown to scale.

Cryo-EM reveals details of the antitermination mechanism. This figure contains structural models generated from cryo-EM data on TECs (PDB IDs: 5MS0 and 6FLQ). (A) The λN antitermination complex (PDB ID: 5MS0) comprising λN (black), NusA (magenta), NusB (red), S10 (orange), NusG (green), and boxA/B RNA (blue), in addition to RNAP (gray). (B) A zoom-in on the boxA/B and λN complex shows extended binding of the nut RNA sequence with multiple protein components, with boxA bound to the NusB-S10 dimer and the boxB hairpin bound to λN and NusA. (C) Formation of the λN antitermination complex shifts the position of NusA (magenta) by 40° away from the RNA exit channel, as compared to NusA (purple) in a TEC constructed with the E. coli his hairpin-mediated pause sequence (PDB ID: 6FLQ). Nascent RNA is shown in green.

RNA elements involved in PA. This figure shows the sequence and secondary structure of RNA elements known or predicted to be utilized in PA mechanisms. Shown are the boxA and boxB elements forming the λN nut sequence as well as rRNA antitermination signal, the put RNA element from phage HK022, EAR from the B. subtilis exopolysaccharide pathway, and a UNCG-type hairpin implicated in LoaP antitermination.

Genomic context of NusG paralog antitermination systems. This figure illustrates the transcripts regulated by the RfaH, LoaP, and UpxY antitermination systems. (A) RfaH regulates multiple pathways in E. coli, including the hemolysin, F pilus, and lipo- and exopolysaccharide operons. Each regulated transcript includes the DNA ops element for RfaH recruitment. RfaH promotes antitermination of Rho-dependent promoters. (B) LoaP regulates two polyketide antibiotic operons in B. velezensis: the dfn difficidin operon and the mln macrolactin operon. LoaP is found divergently oriented upstream of the dfn operon. Each transcript includes a required sequence region in the 5′ leader region, which might include a functionally important hairpin followed by an intrinsic terminator. Additional intrinsic terminator sites have been implicated within the dfn and mln operons, although they are not shown in this figure. (C) UpxY proteins regulate multiple capsular polysaccharide pathways in B. fragilis. Each polysaccharide operon includes both a UpxY and UpxZ protein involved in targeted regulation, with the 5′ leader sequence required for antitermination. B. fragilis has eight distinct polysaccharide operons containing UpxY proteins. Gray rectangles represent multigene operons. RNA elements potentially involved in antitermination are shown in blue, and proteins and protein-coding genes involved in antitermination are shown in green.

The RfaH CTD undergoes a large conformational shift from an α-helix to a β-barrel. Full-length RfaH (left) exists as an autoinhibited structure with the CTD (blue) in an α-helix conformation bound to the NTD (red) (PDB ID: 2OUG). Upon binding to RNAP and the ops DNA, the CTD (right) is released and forms the β-barrel conformation characteristic of NusG KOW domains (PDB ID: 2LCL).

Phylogenetic tree of NusG, Spt5, and specialized NusG paralog groups. Represented NusG sequences selected from the subgroups discussed in this review form the NusG family. Bacterial sequences from core NusG proteins are found in all bacteria, while a variety of paralogs are found in diverse bacteria phyla. Some groups of NusG paralogs are commonly found in or adjacent to large gene clusters coding for production of polysaccharides (PS) or polyketides (PK).