In vitro approaches to analysis of transcription termination

Abstract

Transcription termination is an important event in the transcription cycle that has been exploited in a variety of genetic regulatory mechanisms. Analysis of transcription termination is greatly facilitated by in vitro approaches. We describe a basic protocol for analysis of transcription termination in vitro, and include descriptions of parameters that can be modified for specific types of experimental questions.

Fig. 1

Single round termination assay design. (a) General features of the template. The template should encode a strong promoter (with -35 and -10 hexamers elements recognized by primary σ factors), a good initial transcribed sequence (ITS, at least 20 nt long and missing one of the bases), and a region of interest. For linear templates, at least 50 bp of DNA upstream of the transcription start site (a bent arrow, +1) and at least 30 bp downstream from the studied termination site(s) should be included to allow for proper recognition and for differentiation of terminated and readthrough products. (b) Examples of promoter-ITS combinations. The sequence of the non-template strand is shown, the RNA sequence is the same but with Us in place of Ts). pIA171 [32] has a phage T7A1 promoter followed by a 29 nt long T-less region; pIA226 [32] has a λ P_R promoter and a 26 nt long C-less region. (c) Assay design (for pIA171). This protocol is suitable for RNAPs from E. coli or other mesophilic species; for RNAPs from thermophilic organisms (e.g., Thermus), the initiation reaction must be carried out at 55°C. Halted complexes will be radiolabeled in the 5′ end at several positions (10 if α-[³²P]-GTP is used with pIA226-derived template).

Fig. 2

Differential effect of NusG protein on termination at three representative intrinsic terminators. (a) Template structure. All three templates share the λ P_R promoter and the 26 nt C-less ITS; the downstream regions differ and each encodes an RNA hairpin followed by a U-rich region. The sizes of the terminated and run-off RNA products are shown. (b) Products of the single-round termination reactions. Reactions were carried out as in 2.5) with E. coli RNAP and E. coli NusG (added at 100 nM where indicated). RNA products were separated on a 6% denaturing gel; terminated (T, filled circles) and run-off (RO, open circles) RNAs are indicated for each terminator. The “M” lane was loaded with [³²P]-labeled pBR322 MspI digest, with sizes indicated on the right; note that the RNA species migrate more slowly than the DNA fragments of the same size. (c) Quantification of termination efficiency. Termination efficiency is defined as the fraction of RNA at the terminator (T) relative to the total RNA (which equals the sum of T and RO if no other products are present). The three terminators tested differ in their termination efficiencies as well as in their response to NusG.

Fig. 3

SAM-dependent transcription termination of the S box riboswitch. (a) In vitro transcription of a B. subtilis yitJ template in the presence or absence of SAM. The template consists of the yitJ 5′ sequence (residues +14 to +289, relative to the yitJ transcription start-site) preceded by the constitutive B. subtilis glyQS promoter. Transcription by B. subtilis RNAP was initiated with ApC (corresponding to the +1/+2 positions of the transcript) in the presence of ATP, CTP and UTP to generate a halted TEC at +17. SAM was added as indicated (0-2.4 μM final concentration), and the products were resolved by 6% denaturing PAGE. Terminated (T, 185 nt, filled circles) and run-off (RO, 235 nt, open circles) RNAs are labeled. (b) Quantification of the SAM response. Termination efficiency is defined as the fraction of RNA at the terminator (T) relative to the total RNA (T + RO).