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CH391L/S2013 Hala Ouzon Feb 13 2013
Linking RNA Polymerase Backtracking to Genome Instability in E. coli 
Unlike eukaryotes where translation and transcription take place in different cellular compartments, in bacteria, like E.Coli, the two events are coupled in time and space. During transcription RNA polymerase (RNAp) forms a very stable complex with DNA which is called the elongation complex (EC). This complex is the target of transcriptional regulation in both prokaryotes and eukaryotes. The rate of replication is higher than the rate of transcription which frequently causes collisions between the replisome and RNAp leading to DNA damage. Furthermore EC might slide in the reverse direction along DNA and RNA in an attempt to fix an incorrectly incorporated base which cause the EC to pause and increase the chance of DNA damage. Using in situ chloroacetaldehyde (CAA) DNA footprinting and pulse-field gel electrophoreses (PFGE) , this group shows that codirectional collision between the replisonme and EC (paused or backtracked) damaged DNA. And using these same techniques they showed that this damage is actually double strand breaks (DSB) located at the position of the arrested EC.
PFGE: is used to visualize the small difference between circular E.coli chromosome and linear E.Coli chromosome. PFGE uses a periodic change of field direction which makes different length DNA react to the field at different rates, thus enabling the separation of by size of large fragments of DNA.
In situ DNA footprinting is a method to investigate the sequence specificity of a DNA-binding protein. By using chemical or enzymatic activity, a DNA bound to a protein can be cleaved. DNA fragments can then be run on a gel to determine which bases are protected from attack when the protein is bound. . Authors of this paper used chloroacetaldehyde(CAA), which is a single strand-specific probe. CAA can react with unpaired and unprotected adenines and cytosine. In this experiment cells were treated with CAA then plasmids were extracted and the DNA region was analyzed by primer extension. CAA modified bases are revealed by their ability to terminate the extension of 32P end-labeled primer. To illustrate how the method works (see the figure), at the transcription bubble the DNA helix is unwound over 12-16 bp, with the downstream margin of the transcription bubble at the level of the catalytic center of RNAp. Most of the base residues on the non-transcribed strand are accessible to the single strand-specific probes and thus they are exposed to the solvent. The protection of 8-12 nucleotides of the template strand from single strand-specific chemical probes very often has been taken as an indication of the presence of an extended RNA:DNA hybrid within transcription elongation complexes. Modified bases were detected by primer extension and then by running the extension products on a 6% polyacrylamide gel. 
Glossary of terms
Cl857 is a temperature sensitive repressor allows for activation of pL promoter at 42°C.
λ N codes for an antitermination protein (stimulates elongation), binds to nutL site on RNA
HK022 Nun factors: inhibits elongation and therefore arrests EC, binds to nutL site on RNA 
mfd: transcription-repair coupling factor
RBS: Expression of foreign genes in Escherichia coli requires the combination of prokaryotic transcription and translation elements with a coding region for the foreign gene. Commonly, this results in only modest expression of the foreign gene product. Therefore ribosome-binding site RBS is needed to enhance the translation of foreign genes. 
Rho: a prokaryotic protein involved in termination of transcription.
HU: replication inhibitor.
BCM: antibiotic bicyclomycin, specifically targets and inhibits Rho.
Gre A and GreB elongation factors.
Cm: Translation elongation inhibitor chloramphenicol. (it slows down translation inducing pauses of ECs)
Two plasmids were used, one to monitor codirectional collisions and the other to monitor head-on collisions. Both had the phage λ cl857 pL-nutL-RBSN-UTR cassette with CoIE1 origin of replication. In pCODIR plasmid the phage λ was oriented codirectionally with the origin of replication, whereas the pHDON plasmid had phage λ oriented head-on with origin of replication.
Simulated ECs arrest was done by inducing the pL promoter in wild type and Mfd deficient (∆mfd) E.Coli cells, in the presence and absence of HK022 nun+ prophage. The effect of Nun arrested ECs on the chromosome stability was then asessed by CCA footprinting. Both DNA strands showed the same pattern of DNA breaks regardless of promoter orientation. Mfd relieved codirectional collision DNA damage but did not for the head-on collision DNA damage. If cells were treated with replication inhibitor (HU) or if the promoter was not induced no DNA breaks was detected in either plasmid. These results showed that both collisions between Nun-induced ECs and replisome induced DSB and Mfn actually cannot release Nun-arrested ECs in head-on collision. These findings are contradict a previous study that stated otherwise.
To test whether spontaneously arrested ECs also interfere with replication and cause DSB, they manipulated the rate of RNAp backtracking using the GreA and GreB, enzymes used in cells to minimize backtracking. These enzymes reactivate ECs by by stimulating the formation of a new 3’OH terminus. To demonstrate the role of GreA and GreB as anti-backtracking machinery and to see the anti-backtracking effect on genome stability, this group noticed that when deleting greA and greB a big cluster of DSB around 30 nucleotides downstream of RBS in pCODIR is formed (but not for pHDON). These DSBs were dependent on both replication and transcription because they were eliminated in an induced promoter or HU treated cells (a replication inhibitor). The DNA lesion was confirmed to be a DSB by running on a gel and monitoring the fast migration of the single stranded plasmid compared to the circular one. Therefore this group was able to conclude that naturally backtracked ECs cause DSBs due to codirectional collisions with replisome. They also demonstrated the important role the ribosome (i,e translation) plays in suppressing RNAp backtracking and by that eliminating DSBs: conversion of the untranslated region (UTR) to open reading frame (ORF) eliminated the effect of GreA/B deficiency because of the “pushing” effect of the ribosome on RNAp. The location of the DSB was determined by comparing footprints of the transcription bubble with those of DSB and found that the pattern was very similar which means the position of the DSB matches the position of the Nun-arrested ECs. To confirm that EC indeed can backtrack, the authors cloned and purified a 6His-taged RNAp and let it transcribe a DNA sequence containing pL-nut-RBS-UTR to find that EC actually halted at position +298. When they removed free dNTP and this DNA-RNAp complex was treated with GreA or B, the size of Gre cleavage product was consistent with backtracking of about 15 nucleotides.
Why do we get DSB upon transcription-replication collision? When the EC and replisome collide the RNAp might dislodge leaving the elongated RNA to anneal with the DNA forming an RNA: DNA hybrid which is called the R loop. In the case of backtracking ECs this loop might be large and therefore more stable. In the case of codirectional collision, these R loops provide a 3’OH terminus that could serve as primer for DNA replication. This switch from transcription to replication leads to a break in the leading strand that if not fixed could lead to DSB. To insure that the R loop formation indeed is the cause of DSB, pCODIR, Gre deficient cells were transformed with physiological level RNase H expression and high RNase expression. Only at high RNase expression were DSBs significantly reduced, from which the authors conclude that R loops are the cause of DSBs and that physiological expression levels of RNase are not enough to eliminate the R loops when the anti-backtracking mechanisms are compromised. In support of this mechanism, mutation in RNAp that forms a less stable complex with the DNA and therefore reduces the collisions with replisomes, as well as one that the RNAp ignore an arrest site, like rpoB*35, also suppress the formation of DSB because they avoid the pausing and the backtracking of RNAp.
Do anti-backtracking mechanisms contribute to genome stability? Treating the cells with sub-lethal concentration of Cm slows down the ribosome. This is predicted to increase backtracking and as a consequence increase DSB. Similarly treating the cells with BCM is expected to increase DSBs. However treatment of WT cells with Cm or BCM only reduced DSBs slightly. However, treatment of ∆greB cells with Cm or BCM reduced DSBs a lot. Over expression of GreB largely eliminated DSBs. The authors also checked whether these results would also apply to a non-plasmid system (like the E.Coli chromosome). They did that by visualizing DSBs using pulse-field gel electrophoresis and saw two bands. One band corresponding to the intact circular DNA stays at origin, and the other that corresponds to linear DNA (because of DSB) is seen in the middle of the gel. DNA isolated from WT and ∆gre cells stayed at the origin. DNA isolated form ∆gre, Cm treated cells showed DSBs, these DSBs were eliminated with high expression of RNase or rpoB*35 allele. Monitoring the SOS response with recA-gfp fusion was another way to monitor the anti-backtracking effect on genome stability. Treating cells with Cm induced SOS response (fluorescence). ∆gre cells treated with Cm gave an even higher fluorescence signal. And treating ∆gre cells with Cm and BCM, at a concentration that had almost no effect when administered by itself, induced a yet higher fluorescence signal. GreB over expression suppressed hyperinduction of SOS response by Cm.
The ribosome is the primarily sensor of cellular metabolism and various stresses. In this study we see how factors that affect the ribosome movement affect genome stability. We can see how modulating the rate of ribosome movement by rare codons, for example, can effect genome stability. When translation in insufficient, cells utilize different mechanisms to prevent ECs backtracking such as: 1) Rho factor, that suppress backtracking by terminating transcription, 2) Mfd, which disrupt ECs arrest, and 3) GreA/GreB factors, which suppress DSBs by restarting backtracked ECs.
1. Dutta, D., et al., Linking RNA polymerase backtracking to genome instability in E. coli. Cell, 2011. 146(4): p. 533-43.
2. Korzheva, N. and A. Mustaev, Transcription elongation complex: structure and function. Curr Opin Microbiol, 2001. 4(2): p. 119-25.
3. Shaevitz, J.W., et al., Backtracking by single RNA polymerase molecules observed at near-base-pair resolution. Nature, 2003. 426(6967): p. 684-7.
4. Guerin, M., M. Leng, and A.R. Rahmouni, High resolution mapping of E.coli transcription elongation complex in situ reveals protein interactions with the non-transcribed strand. EMBO J, 1996. 15(19): p. 5397-407.
5. Proshkin, S., et al., Cooperation between translating ribosomes and RNA polymerase in transcription elongation. Science, 2010. 328(5977): p. 504-8.
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