Elongation/Termination: First, an overview of what hold RNAP on DNA during elongation Then some examples of control.

1. Elongation/Termination: First, an overview of what hold RNAP on DNA during elongation Then some examples of control. Note: after sigma leaves nusA and sometimes other factors can join. So far, it is not clear exactly how they are involved in termination control.

2. This is a model of the transcription complex in the process of elongation. (The nucleic acid data comes partlyfrom footprinting and cross-linking) Transcription in this direction Rudder helping to keep template DNA melted. 9 bp DNA-RNA hybrid RNA exit channel, covered by flap Blue residues - basic Red residues - acidic A melted base pair is matched to the NTP. The NTP is added to the 3’ end of the RNA. The polymerase ratchets forward; One bp closes behind and a new one opens ahead.

3. Elongation is processive, but polymerase sometimes pauses. When polymerase pauses it has the potential to terminate transcription This only occurs when the RNA is released terminator Pause sites promoter Time after starting a round of transcription

4. These may be paused because the 3’ end of the RNA has ‘slipped out” of the enzyme active site. Footprints at pause sites These footprints show that the elongation complex does not always move smoothly down the DNA during elongation. As RNA synthesis occurs in these extreme examples the polymerase “front end’ appears not to have moved. Footprinting results on stalled complexes from studies of Krummel and Chamberlin [71 and 72]. The DNase I and KMnO4 footprints of complexes stalled at positions 20 (top), 23 (middle) and 27 (bottom). The sequence of the DNA template is shown with the sequence of the transcript below it. The nucleotides on the template and nontemplate strands that were protected from DNase I digestion are shown as bars above and below the DNA sequence. The region that was susceptible to KMnO4, and therefore is single stranded, is shown in the gray box. An underlined "T" indicates that thymine residue was susceptible to KMnO4. The data are taken from the PhD thesis of Krummel [97].

5. Pausing is probably due to misalignment between the RNA 3' end and the active site. How DNA or RNA or hybrid sequences cause this is not known. bind NTP chemistry ratchet RNA Smooth elongation RNA end Misalignment during elongation - backtracking observed frequently Active site (BBA) - Gene Structure and Expression Volume 1577, Issue 2 , 13 September 2002, Pages 224-239

6. Backtracked RNAPs can be re-activated by Gre factors Recall that Gre factors enter secondary channel and position acidic residues at active site of RNAP. This stabilizes a second Mg setting up metal-catalyzed cleavage of the RNA (see next slide). cleavage re-aligns active center with the 3’ end of the RNA. Elongation can resume. Opalaka…Darst - Cell. 2003 Aug 8;114(3):335-4

7. Evidence for Gre-induced cleavage via acidic residues at active site of RNAP RNA from stalled RNAP released cleavage products mutants inhibit cleavage Other experiments show that GreB increases elongation rate and suppresses pausing

8. RNAP also transiently stalls and backtracks at roadblocks (may be common on real chromosomes) Experiment: Block with stuck R1 at limiting RNAP. Wash away excess free RNAP. Observe backtracking by gre-induced shortened RNA (80-long). Remove roadblock with salt and RNAP can re-position (resistant 84-long) EMBO J. 2003 Sep 15;22(18):4719-2 Note: claims that re-activation is assisted by trailing RNAPs - check if interested

9. Termination will occur when RNA is released during a pause. When polymerase pauses it has the potential to terminate transcription This only occurs when the RNA is released terminator Pause sites Question: What holds the RNA in the elongation complex? promoter Time after starting a round of transcription

10. 3 interactions with polymerase keep the RNA in the elongation complex. In addition the RNA is bound to the DNA in the hybrid. Hybrid binding site 2 RNA binding sites Exit channel

11. What are the signals associated with terminators? Generally there are 2 types: A GC-rich partial dyad followed by a stretch that codes for oligo uridine. The U-stretch is at termination site. The dyad is just upstream. These signals work primarily through RNA rather than DNA. TTTTTT AAAAAA DNA RNA UUUUUU Terminator hairpin U stretch (there also may be an influence of DNA just downstream from the U-stretch.) How do these work to de-stabilize the RNA?

12. Here's the TEC at a potential terminator: RNA hairpin can form here at a pause site. Hairpin RNA formation diminishes RNAP affinity for RNA. Therefore, when the hybrid transiently dissociates during the pause, the RNA is released and termination occurs The rU:dA hybrid is the weakest of all possible hybrids. Hybrid binding site is rU:dA RNA binding sites

13. Another view of termination This state binds the RNA weakly and releases it --- termination Figure 1 The mechanism of intrinsic termination. Termination begins with a brief pause (1.5-2 s) at the termination point (e.g. the U7 position of the tR2 terminator) induced by the T-stretch of the terminator. Three RNAP nucleic acid binding sites are indicated in the paused complex: HBS (RNA:DNA hybrid binding site), RBS ('tight' RNA-binding site), and UBS ('weak' upstream RNA-binding site) Transition from a paused to a termination complex occurs due to hairpin formation and involves breaking protein-RNA contacts in the UBS and the RBS, partial breaking of contacts in the HBS due to unwinding of 5-6 bp of the hybrid, irreversible complex inactivation, and rewinding of the transcription bubble from behind. NusA stimulates hairpin formation principally by weakening the contacts between the potential ascending loop of the hairpin (the 'upstream shoulder' of the unfolded hairpin) and the UBS, and to a lesser extent by prolonging pausing at the termination point

14. Another signal for termination is the absence of RNA secondary structure. In this case the RNA helicase rho binds the RNA and releases it. Rho is a hexamer ring that can load ssRNA, if it is available. Most terminators are downstream from the translation stop codon. This is to allow increased access of rho to the RNA at this time. at the end of a gene When rho approaches polymerase, it this thought that the ring closes and then the ATPase pulls out the RNA. Anti-termination factors can block rho binding to RNA

15. controling metabolites Hide or promote formation of the hairpin readthrough terminate The regulators are quite varied and include the metabolites themselves, proteins, RNA and RNA:protein complexes Control of termination is very simple: Factors control whether the terminator hairpin forms.

16. Examples of regulators: Trp operon (makes trp): When trp is present the hairpin forms (do not want more trp made). When trp absent ribosomes stall and melt the terminator (get transcription now) Regulator proteins bind directly and control hairpin formation. (example coming). FMN, lysine etc. bind RNA directly and induce formation of the terminator hairpin. This prevents unnecessary expression of synthetic enzymes. First overview of FMN and then lysine experiments.

17. FMN will make this form FMN example Terminator hairpin promoter This operon produces proteins needed for synthesis of FMN If enough FMN If not enough FMN FMN causes the terminator hairpin to form as shown in the next slide. Remarkably, FMN binds RNA and stabilizes a structure that exposes the hairpin

18. Termination by FMN + FMN - terminator hairpin forms No FMN - structure forms without terminator hairpin. Therefore, terminate transcription only when FMN is present so don’t make proteins to synthesize more.

19. Examples of termination structures stabilized by metabolites. All allow formation of the terminator hairpin. (normally the hairpin elements are tied up in more stable structures).

20. Forms when lys is present L-box sequences prevent biosynthesis of lysine when it is already present. PNAS | October 14, 2003 | vol. 100 | no. 21 | 12057-12062 - B. subtilis system Terminator hairpin promoter This operon produces proteins needed for synthesis of lys If enough lys If not enough lys

21. ReadThrough Termination Changes in RNA structure can alter termination.

22. Lysine binds RNA directly and leads to less digestion (red), defining its binding site in the predicted structure GENES & DEVELOPMENT 17:2688-2697, 2003

23. The lys-bound structure allows a terminator hairpin to form M1 de-stabilizes terminator hairpin and allows more readthrough M2 decreases stability of anti-loop. Get more termination without lysine. M3 restores stability lost in M2. A bit closer to normal regulation.

24. Riboswitches are very common in gram-positive bacteria and other organisms. Some think this is the oldest type of regulation because it can use direct binding of metabolites and RNA.

25. The protein binds here - next slide Some systems using anti-terminator proteins The protein family. They bind and block formation of terminator hairpins. The RNA targets (anti-terminators). The line indicates nucleotides needed by the terminator hairpin. The EMBO Journal, Vol. 21, No. 8 pp. 1987-1997, 2002

26. The protein stabilizes base anti- forms and blocks formation of a terminator hairpin. Therefore it triggers transcription of genes needed under these conditions.

27. That’s it. Questions about the exam?