Genetics 304 Lecture 6

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1 Genetics 304 Lecture 6 00/01/27 Assigned Readings Busby, S. and R.H. Ebright (1994). Promoter structure, promoter recognition, and transcription activation in prokaryotes. Cell 79: Reed, W.L. and R.F. Schleif (1999). Hemiplegic mutations in AraC protein. J. Mol. Biol. 294: Supplementary Reading Gusarov, I. and E. Nudler (1999). The mechanism of intrinsic transcription termination. Mol. Cell 3: How is transcription terminated? -protein RNA interactions are important -the ability of the RNAP to terminate is established near or at the RP I RP E step of transcription initiation. RNAP RNAP RNAP core holoenzyme RP E Sigma factor Termination competency subunit, e.g. NusA 2 general mechanisms of transcription termination in prokaryotes (1) intrinsic termination sequence that has within itself the information to cause termination (2) rho-dependent termination requires the protein Rho Intrinsic terminators -most common in prokaryotes (phage usually use Rho) -very few genes/operons use rho-dependent termination in E. coli, however a mutation in rho is lethal, therefore it is an important mechanism -how do you find a termination sequence? Determine how long the transcript is (i.e. Northern analysis), then look at the DNA sequences that far from the start site of transcription. -found G-C rich hairpin loops followed by runs of U s in the RNA (template run of A s) 31

2 G/C rich UUUUUUU Experiments in vitro mutagenesis -substitutions of bases in the neck of the hairpin then determine rate of termination in comparison to wildtype sequence -switch G-C pair to a C-G pair - 100% 85% termination -switch a different G-C pair to a G-G pair 100% 5% termination -switch G-C pair to a U-A pair 100% 40% termination -differences are seen with where the base changes are made in the hairpin -anything less than 6 U s did not terminate well -conclusions from in vitro experiments (1) all intrinsic promoters use this general structure (2) not all hairpins are terminators (3) the run of U s is essential for termination Models of how this works (1) competitive kinetic model -formation of the hairpin loop in RNA caused pausing increased probability that RNAP will dissociate, suggesting that there is a constant equilibrium between RNAP elongation and RNAP dissociation. This equilibrium can be skewed towards dissociation by the intrinsic terminator. (2) Allosteric model -there are contacts between the RNA and RNAP -formation of the termination structure causes a conformation change in the RNAP that loosens contacts between RNA/template and RNAP Recent studies -magnetic resonance imaging under various conditions of termination -RNAP contact points that stabilize its structure a. double strand DNA (dsdna) binding site (DBS) b. RNA/DNA (hybrid) binding site (HBS) c. Single strand RNA binding site (RBS) -all three of these binding sites are important in maintaining structure of RNAP elongating complex without termination, this complex is very stable. Cross-linking studies -links RNA-protein, RNA-DNA, DNA-protein -able to probe where the physical contacts are -RNAP pauses at run of U s during this pause the hairpin forms -TEC = ternary elongation complex = RP E 32

3 TEC paused TEC trapped TEC -the loop destabilizes the RBS, which destabilizes HBS causes RNAP dissociation -any process that leads to anti-termination has to act before or at this pausing step rho-dependent termination -discovered biochemically -rho is an essential gene -Rho functions as a hexamer (figure from text 11.27) RNA Hexamer of Rho subunits rolls along RNA -important RNA sequences that allow rho-dependent termination -an accessible C-rich, G-poor region in the RNA preceding the termination site -site of termination associated with an A/T rich template Hot-pursuit model -Rho uses ATP to move ( roll ) along DNA -Rho has ATP-dependent helicase activity -once Rho has bound to the transcript, it can move faster than RNAP -Rho uses its helicase activity to destabilize RNA-DNA hybrid region HBS destabilized -Why doesn t rho-dependent termination happen all the time, these sequences are not uncommon? -coupled transcription-translation of mrna in prokaryotes -ribosomes bound to the mrna block Rho from binding and moving along the RNA -after the ribosomes fall off (i.e. translation termination) Rho is able to access the RNAP 33

4 An old class of mutations, polar mutations, can be explained by this model -in polar mutants, transcripts terminate prematurely, such that a mutation in a gene of an operon leads to loss of expression of all following genes in the operon A B C WT A B C Polar mutants * - stop codon -the presence of a stop codon in the first gene of the operon causes translation termination early, which leads to early termination of transcription -these mutant phenotypes could be suppressed by non-sense suppressors Figure from text. -the ribosome falls off when it encounters the stop codon - region of transcript that becomes accessible to Rho that normally is not accessible. If Rho binding and termination sequences are present RNAP dissociates -under normal circumstances, the distance or sequence between the stop codon and Shine- Delgarno sequence of two genes in an operon is not sufficient to allow Rho binding Poly-cistronic messages that do not get translated how do these deal with rho-dependent termination? -e.g. rrn operon -composed of rrna and trna encoding genes -7 copies of the rrn operon in the E. coli genome 34

5 -there is a short motif in all 7 operons immediately after the start site -box A motif -reporter construct made to test whether or not this box A allows anti-termination Box A CAT Rho-dep. termination sequence -CAT = chloramphenicol acetyl transferase -box A does not allow rho-dependent termination -mutations could be made in the box A that dissabled this function and allowed termination Anti-termination factors (prevent rho-dependent termination) -S10 ribosomal protein (appears to have two distinct functions) -NusB -these 10 proteins form a complex that binds to RNAP in the context of transcription of a box A sequence. -beyond the box A, the RP E -S10-NusB complex is resistant to Rho. 35