The functional anatomy of an intrinsic transcription terminator

Abstract

To induce dissociation of the transcription elongation complex, a typical intrinsic terminator forms a G.C-rich hairpin structure upstream from a U-rich run of approximately eight nucleotides that define the transcript 3' end. Here, we have adapted the nucleotide analog interference mapping (NAIM) approach to identify the critical RNA atoms and functional groups of an intrinsic terminator during transcription with T7 RNA polymerase. The results show that discrete components within the lower half of the hairpin stem form transient termination-specific contacts with the RNA polymerase. Moreover, disruption of interactions with backbone components of the transcript region hybridized to the DNA template favors termination. Importantly, comparative NAIM of termination events occurring at consecutive positions revealed overlapping but distinct sets of functionally important residues. Altogether, the data identify a collection of RNA terminator components, interactions and spacing constraints that govern efficient transcript release. The results also suggest specific architectural rearrangements of the transcription complex that may participate in allosteric control of intrinsic transcription termination.

Fig. 1. Transcription termination with wild-type (WT) and mutant (Y639F) T7 RNAPs. (A) The run-off (RO) and termination (Ter) products obtained with linearized plasmids pAST7-T1 (T1), pMBT7-trpA (trpA) and pMBT7-thR (thR) were resolved on 9% denaturing poly acrylamide gels. Global termination efficiencies (TEs) are indicated below the lanes. Individual TEs at the minor release site (T₊₁) of the T1 terminator are enclosed in parentheses. (B) Fractionation of a transcription reaction with Y639F RNAP (lane C) on 100 kDa Microcon columns before (lanes Native) or after (lanes PhOH) phenol extraction and ethanol precipitation. Lanes R and E refer to the membrane retenate and eluate fractions, respectively. (C) Identification of transcript 3′ ends. Phosphorothioate sequencing was performed on 3′-end-labeled T transcripts as described previously (Boudvillain et al., 2002). Minor T₊₁ transcripts with labeled 3′ ends could not be prepared in sufficient amount for direct comparison.

Fig. 2. NAIM of the transcripts released at the major site (T) of the T1 terminator. Numbering of the nucleotide positions is from the +1 start site of transcription. (A) Iodine sequencing of termination (T) and run-off (R) products of transcription reactions containing either ATPαS (AαS) or 7-deaza-ATPαS (C7-A). The positions of NAIM effects are indicated by arrows. (B) The identification of interference effects (arrows) using the discrimination factor λ (see Materials and methods) is presented for the Rp-phosphorothioate (αS) and 2′-deoxy (dNαS) incorporations. Dotted lines correspond to the cut-offs (±2.5) that were applied to select statistically significant NAIM effects.

Fig. 3. Summary of the NαS effects on termination at the (A) T site; (B) T₊₁ site. Only the NαS analogs that yielded interference signals are shown (key is inset). The positions of NαS effects are indicated on the secondary structure of the transcripts (upstream transcript positions did not exhibit NαS effects and are not shown). The levels of NαS incorporations could not be quantified accurately for the last 3′-nucleotide of the transcripts (in italics). The key to color coding of weak, moderate and strong effects is shown above the sequence of the T transcript. (C) Comparison of NαS effects on termination at the T and T₊₁ sites using the discrimination factor λ (see Materials and methods). The dashed line is the best linear fit of the data points (y intercept = 0.07; slope = 0.67). Dark and light gray regions contain NαS modifications selectively affecting termination at the T and T₊₁ sites, respectively. Arrows identify IαS and dNαS effects at positions 59, 83 and 85. The number of hits per graph region is depicted schematically below the diagram.

Fig. 4. Comparative NAIM of the major (T) and minor (T₊₁) T1 termination products. Representative sequencing gels showing Rp- phosphorothioate (NαS), 2′-deoxy (dNαS), 7-deaza-GαS (C7-G) and inosine-αS (IαS) modification effects. Lanes R refer to the run-off transcripts.

Fig. 5. RNAP contacts to the 3′ tips of transcripts. An enlarged view of a gel showing UαS and dUαS signals at 3′-terminal positions of the T and T₊₁ transcripts. The dUαS region is also shown with a lower gel exposure. Gel migration has been increased to improve the resolution of the 3′ region (compare with Figure 4). Gel lanes are annotated as in Figure 4. Strong termination-reducing dUαS effects at the transcript 3′ tips are indicated by black triangles.

Fig. 6. Steric clashes and interactions between a terminator RNA and important components of T7 RNAP. (A) The transcript 3′ end (in blue), and Y639 (orange), D537 (pink) and D812 (red) RNAP side chains in the active site of a T7 TEC (Yin and Steitz, 2002). The DNA template is in brown and the 2′-OH group of the 3′-ribonucleotide is in yellow. (B) Top: superimposition of the RNA transcript and RNAP interacting residues from two different T7 TEC structures (PDB 1MSW and 1H38 are orange and blue, respectively). The 2′-hydroxyls are green, except for the one of the 3′-ribo nucleotide, which is yellow. Bottom: hydrophobic RNAP residues are shown in gray and polar/charged residues in purple. (C) Docking of a 22 nucleotide hairpin (PDB: 1F9L) similar to the T1 hairpin (same loop but slightly different stem sequence) into the RNA exit channel of the T7 TEC (PDB: 1MSW) using the DeepView program (Guex and Peitsch, 1997). The hairpin 3′ end and the 5′ end of the seventh nucleotide of the TEC transcript have been linked manually. None of the various orientations of the hairpin that have been tried with respect to the TEC were devoid of steric clashes between the RNAP and exiting RNA. Because our primary intent was to provide plausible and general information about the respective positions of terminator and RNAP components and because the T7 TEC is likely to undergo substantial conformational rearrangements upon hairpin formation, we did not attempt further model optimization. The first 5 bp of the terminator hairpin that contain strong NαS effects are red. Other RNA residues are yellow; DNA strands are brown. Major RNAP mobile substructures (Tahirov et al., 2002; Yin and Steitz, 2002) are colored as follows: specificity loop (739–772) in green, flap domain (152–205) in blue, C-linker (258–266) in cyan, N-subdomain (2–71) in magenta. The transcript region upstream from the terminator hairpin is not represented. The figure has been prepared with Protein Explorer 1.98 (http://proteinexplorer.org).