Factor-stimulated intrinsic termination: getting by with a little help from some friends

Abstract

Transcription termination is known to occur via two mechanisms in bacteria, intrinsic termination (also frequently referred to as Rho-independent or factor-independent termination) and Rho-dependent termination. Based primarily on in vitro studies using Escherichia coli RNA polymerase, it was generally assumed that intrinsic termination and Rho-dependent termination are distinct mechanisms and that the signals required for intrinsic termination are present primarily within the nucleic acids. In this review, we detail recent findings from studies in Bacillus subtilis showing that intrinsic termination in this organism is highly stimulated by NusA, NusG, and even Rho. In NusA-stimulated intrinsic termination, NusA facilitates the formation of weak terminator hairpins and compensates for distal U-rich tract interruptions. In NusG-stimulated intrinsic termination, NusG stabilizes a sequence-dependent pause at the point of termination, which extends the time frame for RNA hairpins with weak terminal base pairs to form in either a NusA-stimulated or a NusA-independent fashion. In Rho-stimulated intrinsic termination, Rho prevents the formation of antiterminator-like RNA structures that could otherwise compete with the terminator hairpin. Combined, NusA, NusG, and Rho stimulate approximately 97% of all intrinsic terminators in B. subtilis. Thus, the general view that intrinsic termination is primarily a factor-independent process needs to be revised to account for recent findings. Moreover, the historical distinction between Rho-dependent and intrinsic termination is overly simplistic and needs to be modernized.

Keywords: Intrinsic termination; NusA; NusG; Rho; Rho-dependent termination.

Figure 1.

Term-seq and a model of NusA-stimulated intrinsic termination. a. Schematic of Term-seq. The protocol starts with growing a bacterial culture to a certain cell density, after which total RNA (green) is extracted and then depleted of rRNA. A unique oligo (red) is ligated to the 3’ end of each isolated transcript to preserve the authentic 3’ end without altering the natural biological sequence (green). These RNAs are then subjected to short-read sequencing on the illumina platform. The sequencing reads provide both RNA-seq and 3’ end information. b. A model of NusA-stimulated intrinsic termination. In this model, NusA (yellow) is bound to RNAP (light blue) during transcription elongation. The DNA, RNA-DNA hybrid, and the nascent RNA is shown. NusA assists intrinsic termination of terminators with bulges in the hairpin (red portion of hairpin) and distal U-rich tract interruptions (red base pairs in the RNA:DNA hybrid).

Figure 2.

Model of NusG-stimulated intrinsic termination. In this model, NusG (purple) is bound to RNAP (light blue) during transcription elongation. The DNA, RNA-DNA hybrid, and the nascent RNA is shown. NusG can be seen making contacts with the non-template DNA strand at the positions indicated by the flipped-out bases. NusG assists intrinsic termination of terminators with terminal A-U and/or G-U base pairs (red base pairs in the terminator hairpin) and distal U-rich tract interruptions (red base pairs in RNA:DNA hybrid).

Figure 3.

Models of Rho-stimulated intrinsic termination and hybrid Rho-dependent termination. a. Model of Rho-stimulated intrinsic termination. Model on the left illustrates transcription when Rho is not contacting RNAP (light blue). In this case an AT-like structure forms outside of the RNA exit channel. Model on the right illustrates transcription when Rho (green) is contacting RNAP. In this case a terminator hairpin forms in the RNA exit channel. The DNA is dark blue and the RNA is dark green. A competing AT-like structure can be seen forming when Rho is not present. The intrinsic terminator hairpin can be seen forming when Rho is present. The red portion of these two hairpins corresponds to the same segment of the nascent transcript. Rho can be seen contacting the nascent transcript upstream of the intrinsic terminator hairpin, thereby preventing the formation of the competing structure. b. Model of hybrid Rho-dependent termination. From left to right. The TEC at an intrinsic terminator. Rho can be seen stimulating an intrinsic terminator. Transcript release is depicted by the upward pointing arrow. In cases where readthrough occurs, a rut site (red) is encoded shortly downstream of the intrinsic terminator sequence. Once transcription has proceeded past the rut site, Rho contacts the rut site and stimulates classical Rho-dependent termination across a broad window, depicted by multiple upward pointing arrows.

Figure 4.

A holistic model of intrinsic termination. Model on the left illustrates transcription of the intrinsic terminator sequence when no transcription factors are contacting RNAP (light blue). Model on the right illustrates transcription of the intrinsic terminator sequence when NusA (yellow), NusG (purple), and Rho (green) are contacting RNAP. The DNA is dark blue and the RNA is dark green. A competing AT-like structure can be seen forming when transcription factors are absent. The intrinsic terminator hairpin can be seen forming when transcription factors are present. The red portion of these two hairpin strands corresponds to the same segment of the nascent transcript. Rho can be seen contacting the nascent transcript upstream of the intrinsic terminator hairpin, thereby preventing the formation of the competing AT-like structure. NusG can be seen contacting Rho. While not shown explicitly here, it should be noted that NusA has also been reported to physically contact Rho within the E. coli TEC [10,11]. In addition, NusG can be seen making contacts with the non-template DNA strand at the positions indicated by the flipped-out bases. Combined, NusA, NusG, and Rho assist intrinsic termination of terminators with terminal A-U and/or G-U base pairs (red base pairs in the terminator hairpin), distal U-rich tract interruptions (red base pairs in RNA:DNA hybrid), and bulges in the hairpin stem.