The antitermination activity of bacteriophage lambda N protein is controlled by the kinetics of an RNA-looping-facilitated interaction with the transcription complex

Abstract

Protein N of bacteriophage lambda activates the lytic phase of phage development in infected Escherichia coli cells by suppressing the activity of transcriptional terminators that prevent the synthesis of essential phage proteins. N binds tightly to the boxB RNA hairpin located near the 5' end of the nascent pL and pR transcripts and induces an antitermination response in the RNA polymerase (RNAP) of elongation complexes located at terminators far downstream. Here we test an RNA looping model for this N-dependent "action at a distance" by cleaving the nascent transcript between boxB and RNAP during transcript elongation. Cleavage decreases antitermination, showing that an intact RNA transcript is required to stabilize the interaction of boxB-bound N with RNAP during transcription. In contrast, an antitermination complex that also contains Nus factors retains N-dependent activity after transcript cleavage, suggesting that these host factors further stabilize the N-RNAP interaction. Thus, the binding of N alone to RNAP is controlled by an RNA looping equilibrium, but after formation of the initial RNA loop and in the presence of Nus factors the system no longer equilibrates on the transcription time scale, meaning that the "range" of antitermination activity along the template in the full antitermination system is kinetically controlled by the dissociation rate of the stabilized N-RNAP complex. Theoretical calculations of nucleic acid end-to-end contact probabilities are used to estimate the local concentrations of boxB-bound N at elongation complexes poised at terminators, and are combined with N activity measurements at various boxB-to-terminator distances to obtain an intrinsic affinity (K(d)) of approximately 2 x 10(-5) M for the N-RNAP interaction. This RNA looping approach is extended to include the effects of N binding at nonspecific RNA sites on the transcript and the implications for transcription control in other regulatory systems are discussed.

Figure 1. Transcription templates and reactions

(A) Transcription templates used to measure the effect on N-dependent antitermination activity of the boxB-to-terminator RNA length. All templates contain pL promoter, nutL DNA encoding the boxB RNA sequence (grey box) and the λ tR′ terminator (black box); template sequences are identical except for insertions between nut and tR′. Insertions share the same 5′ sequence and differ in length of the 3′ end. (B) Read-through (RT) and terminated (Term) RNA bands for transcription reactions performed with templates pRB50, pRB200, pRB400 and pRB800 in the presence of increasing concentrations of λN protein.

Figure 2. Effect of template distance between the boxB-N protein binding site and the terminator on N protein-dependent antitermination activity in the absence of accessory proteins

The fraction of full-length RNA transcripts produced by transcriptional readthrough of terminator tR′ for each template in Figure 1 at the indicated concentrations of N protein are plotted as a function of transcript distance (in nts) between the N binding site (boxB RNA) and the target terminator.

Figure 3. Schematic of transcript cleavage experiments

Procedure used to form stalled antitermination complexes and simultaneously re-extend and cleave RNA transcripts during transcription between boxB and the terminator. Asterisks indicate positions of α-³²P ATP incorporation in the RNA transcript. Re-extension of transcripts and cleavage with RNAse H produces five radiolableled RNAs, corresponding to the 5′ cleavage product and pairs of cut and uncut terminated and readthrough transcripts.

Figure 4. A continuous RNA tether is required for functional interaction of N with RNA polymerase

RNAse H cleavage of the transcript between boxB and the terminator site decreases N-dependent antitermination activity. Elongation complexes stalled between boxB and terminator sequences were hybridized to a ssDNA oligonucleotide, supplemented with N protein (200 nM) and re-extended with NTPs in the presence of RNAse H. Partial RNAse H cleavage of the elongating transcripts yielded four RNA chains corresponding to terminated (Term) and full-length readthrough (RT) products of the RNAse H-cut and uncut transcripts, and two shorter RNAs corresponding to unextended stalled complexes (Unextended Stalled EC) and the 5′-product generated by RNAse H cleavage of the transcript (Cut 5′ end). The fraction of RNAse H-cleaved transcripts (Fraction RT, cut) that read through terminators and the fraction of intact transcripts that read through terminators (Fraction RT, uncut) are indicated at the bottom. The times between addition of RNAse H and the NTP-extension mix are indicated at the top of the gel.

Figure 5. Effect of template distance between the boxB-N protein binding site and the terminator on N-dependent antitermination activity in the presence of NusA

The fraction of full-length RNA transcripts produced by transcriptional readthrough of terminator tR′ for each template in Figure 1 at the indicated concentrations of N protein in the presence of 120 nM NusA protein are plotted as a function of transcript distance between boxB and the terminator.

Figure 6. Antitermination 'range' in the presence of N, NusA, NusB, NusE, NusG and transcript-encoded boxB

Products of transcription reactions performed with templates pRB250, pRB200, pRB400 and pRB800 in the presence of indicated concentrations of N and NusA (120 nM), NusB, NusE and NusG proteins (250nM each). In all cases, the fraction of transcripts that ‘read through’ the terminator was >95%.

Figure 7. A continuous RNA tether is not required for functional interaction of N with RNA polymerase in the presence of accessory factors

RNAse H cleavage of the transcript between boxB and the terminator site does not decrease N-dependent antitermination activity in the presence of transcription accessory factors. Experiments were performed as in Figure 3; i.e., elongation complexes stalled between boxB and terminator sequences were hybridized to a ssDNA oligonucleotide, supplemented with N protein (200 nM), NusA (120 nM), and NusB, E and G (250 nM each), and incubated with RNAse H for the indicated times before NTP addition and resumption of transcription. Reactions yielded four RNA chains corresponding to terminated (Term) and full-length readthrough (RT) products of the RNAse H-cut and uncut transcripts, and two shorter RNAs corresponding to unextended stalled complexes (Unextended Stalled EC) and the 5′-product generated by RNAse H cleavage of the transcript (not shown). The fractions of full-length RNA transcripts produced by transcriptional readthrough of terminator tR′ of RNAse H-cleaved (Fraction RT Cut) and intact (Fraction RT Uncut) transcripts, and the times between addition of RNAse H and the NTP-extension mix, are indicated above and below the relevant lanes.

Figure 8. Comparison of experimental and predicted values for terminator readthrough produced by the addition of N protein as a function of RNA length

Experimental measurements from Figure 2 at input concentrations of 50 (black circles) and 100 (white circles) nM N protein were normalized to the total change in terminator read-through due to the addition of N protein; thus the maximum terminator readthrough at 400 nM N (0.77) in Figure 2 corresponds to a Fraction Terminator Readthrough (right y-axis) of 1, and the terminator readthrough in the absence of N (0.11) in Figure 2 corresponds to a Fraction Terminator Readthrough of 0. Predictions of the fraction of elongation complexes that are bound in cis by transcript-tethered N protein (Fraction RNAP bound by N, left y-axis) at 50 (thin solid lines) and 100 (thick solid lines) nM N protein as a function of increasing RNA loop length were generated using the ‘exact’ model described in the Appendix and a tethered equilibrium constant (K_d) of 2 × 10⁻⁵ M. The K_d for the binding of N to boxB was 0.5 nM at the salt concentrations used in the transcription reactions; apparent K_d values for nonspecific binding of N to ssRNA and dsDNA were 100 nM and 135 nM, respectively. Binding constants were measured and nucleic acid concentrations were estimated as described in Materials and Methods.

Figure 9. Effect of nonspecific binding on the local concentration of N, on N binding to RNAP and on terminator read-through as a function of transcript length. A. Local concentrations of N protein in the presence and absence of nonspecific interactions between N, RNA and DNA

Thick line: predicted local concentration of N (bound at boxB) at RNAP (bound at the terminator) as a function of distance between boxB and the terminator in the absence of competition from nonspecific sites. At a total N protein concentration of 100 nM, more that 99% of the boxB sites are bound. Medium line: average local concentration when 100 nM N protein is partitioned between boxB and nonspecific nucleic acid binding sites. Thin line: average local concentration when 100 nM N protein is partitioned between boxB and the nonspecific nucleic acid binding sites when the transcription template has an additional 800 bp dsDNA adjacent to the transcribed region. Complexes of N with nonspecific RNA possess antitermination activity, and increase the fraction of RNAP bound by N; complexes of N with DNA are inactive for antitermination (see text), and act as a “sink” for free N protein B. Fraction of RNAP bound by ‘cis-looped’ N in the presence and absence of nonspecific interactions. Thick line: predicted fraction of RNAP bound to ‘cis-looped’ N-boxB complexes in the absence of nonspecific binding to RNA and DNA. Medium line: fraction of RNAP bound to cis-looped N protein tethered at boxB or at a nonspecific binding site. Thin line: fraction of RNAP bound to cis-looped N protein tethered at boxB or at a nonspecific site when the transcription template contains an additional 800 bp of dsDNA adjacent to the transcribed region. The simulations were performed using the ‘exact’ model described in the Appendix with a total N protein concentration of 100 nM and the same K_d values for N binding to RNAP and nucleic acid as in Figure 8. Unless otherwise noted, the templates used in the simulations had the same number of DNA base pairs outside the transcribed region as the experimental templates depicted in Figure 1 and described in the Materials and Methods.

Figure 10. Model for control of antitermination by the tethered interaction of N protein with elongation complexes. A. The minimal in vitro antitermination complex

N protein binds to the transcript-encoded boxB sequence and loops to contact RNAP; N also binds to nonspecific sites on the transcript. Binding of tethered N-boxB complexes to RNAP equilibrates during transcription, with fast on- and off-rates (arrows) that permit N to sample the changing local concentration provided by the lengthening RNA. The binding of N to RNAP and antitermination activity are controlled by the transcript length-dependent change in local N concentration. B. The 'physiological' antitermination complex consists of N, RNAP, boxB, and the NusA, B, E and G proteins. N binds to boxB and the high local concentration of N provided by the short RNA transcript facilitates initial binding of the N-boxB to the transcription complex. The interaction of the N-boxB complex with RNAP and Nus factors is persistent relative to the time required for transcription between boxB and the terminator. The binding of N to RNAP and the antitermination activity are therefore not sensitive to transcript length, but depend on the slow (relative to transcription) rate of dissociation of Nus-factor-stabilized N from the elongation complex.

Figure 11. Nonspecific binding of N protein in trans to RNA and DNA controls the fraction of N molecules specifically bound at the boxB RNA site available for RNA looping-facilitated binding of N to RNAP. Effect of nonspecific binding and transcript length on the antitermination activity of N protein bound at the boxB RNA site. A. Number of N molecules bound to the nucleic acid framework of the elongation complex as a function of N concentration

Predicted average number of N molecules bound to the RNA and DNA binding sites of elongation complexes produced by in vitro transcription of the 184 nt template pRB2 with RNAP at the terminator at a total input N concentration of 100 nM. Thick line: total N molecules bound to transcript RNA. Thin line: N molecules bound to boxB RNA. Grey line: N molecules bound to nonspecific DNA. Medium line: N molecules bound to nonspecific RNA sites. B. Probability of N-RNA and N-boxB complexes binding to RNAP and inducing antitermination. Probability that individual N-RNA and N-boxB complexes from Figure 11A will bind RNAP and induce antitermination. DNA acts as a ‘sink’ for N protein and N molecules bound nonspecifically to DNA do not compete for RNAP binding. Thick line: total fraction of RNAP molecules bound by N-boxB and N-RNA complexes. Dashed line: fraction of RNAP bound by N-RNA complexes. Thin line: fraction of RNAP bound by N-boxB complexes. Circles: experimental fraction of elongation complexes reading through the terminator. C. Probability of N-RNA and N-boxB complexes binding to RNAP on a long transcript containing a single boxB binding site. Predicted N-RNAP binding on the 984 nt pRB800 transcript. Thick line: total fraction RNAP bound by N. Dashed line: fraction RNAP bound by N-RNA complexes. Thin line: fraction RNAP bound by N-boxB complexes. D. Probability of N-RNA and N-boxB complexes binding to RNAP on a short transcript containing a single boxB binding site. Predicted N-RNAP binding on a hypothetical transcript containing 48 nt between boxB and terminator. The template used in this simulation has the same number of DNA bp outside the transcribed region as the experimental templates. Thick line: total fraction RNAP bound by N. Dashed line: fraction RNAP bound by N-RNA complexes. Thin line: fraction RNAP bound by N-boxB complexes. We used the ‘exact’ predictive model (see Appendix) to calculate the partitioning of N protein onto boxB, RNA, and DNA and the fraction of elongation complexes that read through the terminator due to a looping-facilitated interaction between RNAP and N protein bound to boxB and/or nonspecific sites on the transcript. The model assumes that the N-RNAP interaction equilibrates on the time scale of elongation, that the affinity of boxB- and RNA-bound N proteins to RNAP is equal, and that interaction between RNAP and a molecule of N protein bound to boxB or a nonspecific site on the transcript results in antitermination. The same K_d values for N binding to RNAP, boxB, nonspecific ssRNA sites and nonspecific dsDNA sites were used as in Figure 8.

Figure A1. Comparison of the ‘exact’ and the ‘independent’ models for N protein binding to nucleic acid components of transcription elongation complexes

Calculations of the average number of N protein molecules bound to nucleic acid components of a transcription elongation complex as a function of increasing N concentration were made using the ‘exact’ method described in the Appendix (black lines), and the ‘independent’ model, which treats every potential binding site on the bare transcript and template as an independent species (grey lines). Thin lines: N protein binding at the boxB RNA site. Medium lines: N protein binding at nonspecific ssRNA sites present in the transcript. Dashed lines: N protein binding at nonspecific dsDNA sites present in the transcription template. Thick lines: N protein binding to the RNA transcript (boxB + nonspecific sites).