Termination and antitermination: RNA polymerase runs a stop sign

Abstract

Termination signals induce rapid and irreversible dissociation of the nascent transcript from RNA polymerase. Terminators at the end of genes prevent unintended transcription into the downstream genes, whereas terminators in the upstream regulatory leader regions adjust expression of the structural genes in response to metabolic and environmental signals. Premature termination within an operon leads to potentially deleterious defects in the expression of the downstream genes, but also provides an important surveillance mechanism. This Review discusses the actions of bacterial and phage antiterminators that allow RNA polymerase to override a terminator when the circumstances demand it.

Figure 1. Schematic model of the elongation complex

Core RNA polymerase (RNAP) (in bacteria, a complex composed of an α-dimer, a β-subunit, a β′-subunit and an ω-subunit) is bound to the DNA duplex composed of the template strand (black) and the non-template strand (blue), and the nascent RNA (red). The α-amino-terminal domains (α-NTDs) serve as a scaffold for complex assembly; the α-carboxy-terminal domains (α-CTDs) and ω-subunit play regulatory roles during initiation. The β- and β′-subunits jointly form the active site and make all the contacts to the nucleic acids. The substrate nucleoside 5′-triphosphate (NTP) (bound to a second Mg²⁺ ion) is thought to enter through the secondary channel. 12–14 bp of the DNA are melted in the transcription bubble. The non-template DNA strand is exposed on the surface, where it may interact with regulatory proteins. The nascent RNA is annealed to the template strand to form 8–9 bp of the RNA–DNA hybrid, which is the key determinant of elongation complex stability^,,. The upstream RNA is extruded through the RNA exit channel formed between the β-flap and β′-clamp.

Figure 2. Bacterial termination signals

Intrinsic terminators are composed of a stable RNA hairpin that can extend to within 8 nucleotides of the 3′ end of the RNA and disrupt the upstream edge of the RNA–DNA hybrid. Transcription elongation protein NusA interacts with the nascent RNA near the exit channel and can stimulate termination. At Rho-dependent terminators, a Rho utilization (rut) element (orange) encoded in the nascent RNA binds to Rho. The initial Rho–rut interactions trigger formation of a stable complex in which the Rho hexamer encircles the RNA and translocates towards RNA polymerase (RNAP). Contacts with rut may persist until Rho reaches RNAP at the actual site of RNA release, which may be located far downstream; however, recent data suggest that Rho–rut contacts are lost earlier ^,. The carboxy-terminal domain of NusG binds to Rho and strongly stimulates its activity in vivo and in vitro^,.

Figure 3. Differential folding of a nascent RNA

a | The leader regions of many operons encode RNA elements that can base pair with different segments of the same mRNA. In a simple scenario, an upstream terminator will form and transcription will stop unless the formation of the terminator hairpin is prevented — for example, by an RNA-binding antiterminator protein. In this case, an antiterminator hairpin will form instead. The number and complexity of alternative structures and the regulatory mechanisms that control the fate of the nascent RNA vary greatly among operons, and new regulators remain to be identified. b | Attenuation in the Escherichia coli trp operon. The leader region upstream of the structural trp genes encodes a 14-residue leader peptide (region 1) and several RNA elements that can form alternative secondary structures. Region 3 can base pair with either region 2 (to form an antiterminator hairpin) or region 4 (to form a terminator hairpin). When Trp levels are low, the ribosome stalls at one of two Trp codons (at positions 10 and 11 within the leader peptide), the antiterminator hairpin forms and Trp biosynthesis genes are expressed, leading to an increase in the concentration of Trp. When Trp levels are high, the ribosome advances into region 2 and blocks the antiterminator. The terminator structure forms instead, and RNA polymerase (RNAP) dissociates upstream of the trpE gene.

Figure 4. A model for termination and antitermination

During rapid elongation (top row), the active site of RNA polymerase (RNAP) is optimized for catalysis. After the enzyme has added a nucleotide to the nascent RNA, the enzyme is in the ‘pre-translocated state’ where the 3′ nucleotide of the RNA occupies the downstream half of the active site. The enzyme then translocates along the DNA to form the ‘post-translocated state’, in which the 3′ OH group of the nascent RNA is positioned in the upstream half of the active site, and the incoming nucleoside 5′-triphosphate (NTP) can readily bind to the vacant downstream half-site of the active site and is subsequently incorporated into the RNA. When the RNAP reaches a pause site, the 3′ OH group remains in the pre-translocated configuration, inhibiting binding of the NTP substrate, and the active site undergoes a rearrangement to yield the elemental pause intermediate in which the 3′ OH may be frayed^,. From this intermediate, RNAP can either escape upon addition of another nucleotide or isomerize into a termination complex. Two mechanisms for formation of the termination complex are currently debated. In the first (shearing or hyper-translocation) model, the RNA 3′ end is lost from the active site when the nascent RNA is pulled upstream by Rho or an RNA hairpin or when the RNAP is pushed forward^,,. In the second (allosteric) model, Rho or terminator hairpin formation induces dramatic changes within the complex (indicated by an altered shape of the enzyme) but the 3′ end of the RNA is retained in the active site^,. An antiterminator protein can directly block formation of the termination complex, prevent isomerization into the elemental pause or stabilize the elongation complex against dissociation.

Figure 5. Processive antitermination mechanisms

In each panel, only the RNA polymerase (RNAP) elements implicated in antitermination are shown. a | Antitermination by phage λ protein N requires assembly of a large ribonucleoprotein complex on two RNA elements, boxA and the N utilization (nut) RNA hairpin. N can directly bind the nut hairpin on its own and it allows RNAP to read through a single terminator (left). However, establishing the long-lived termination-resistant modification of RNAP also requires several host Nus proteins (NusA, NusB, 30S ribosomal protein S10 (also known as NusE) and NusG) to stabilize the antiterminator complex through a network of interactions (right). b | Phage λ protein Q recruitment to RNAP requires the Q utilization (qut) DNA element, which overlaps the promoter and directly binds to Q, and a promoter-proximal pause, which is induced by interactions of region 2 of initiation factor-σ with a –10-like promoter element in the transcribed DNA; σ-factor region 4 also interacts with Q (REF. 80). After recruitment, Q turns into an RNAP subunit and modifies the enzyme into a processive state. NusA can stimulate Q activity. c | Antitermination by polymerase utilization (put) RNA is independent of accessory proteins but does require stable binding of put RNA to the enzyme. d | The amino-terminal domain (NTD) of RfaH binds to the non-template DNA in the elongation complex paused at the operon polarity suppressor (ops) site (left), triggering domain dissociation, which in turn unmasks the site that binds to the β′-clamp helices. RfaH remains bound to the elongation complex through the NTD contacts with the β′-clamp helices, β-gate loop and non-template DNA (right). CTD, carboxy-terminal domain.

Figure 6. Targets for processive antiterminators

The β-subunit (light yellow) and β′-subunit (light green) of RNA polymerase (RNAP) are shown in surface representation, with the key elements shown as cartoons. The non-template DNA strand is shown in blue and the template DNA strand in black. The active-site Mg²⁺ ion (magenta) and Zn²⁺ ion (black) in the zinc-finger motif are shown. The amino terminus of the β′-subunit is marked with a green sphere. The target sites for the antitermination factors are shown as transparent ovals. RfaH has two targets, the β′-clamp helices and the β-gate loop. N, phage λ protein N; put, polymerase utilization; Q, phage λ protein Q.