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1.  Temporal Regulation of Gene Expression of the Escherichia coli Bacteriophage phiEco32 
Journal of Molecular Biology  2012;416(3):389-399.
Escherichia coli phage phiEco32 encodes two proteins that bind to host RNA polymerase — gp79, a novel protein, and gp36, a distant homolog of σ70 family proteins. Here, we investigated the temporal pattern of phiEco32 and host gene expression during the infection. Host transcription shut-off and three distinct bacteriophage temporal gene classes – early, middle, and late – were revealed. A combination of bioinformatic and biochemical approaches allowed identification of phage promoters recognized by host RNA polymerase holoenzyme containing the σ70 factor. These promoters are located upstream of early phage genes. A combination of macroarray data, primer extension, and in vitro transcription analyses allowed identification of six promoters recognized by RNA polymerase holoenzyme containing gp36. These promoters are characterized by a single consensus element tAATGTAtA and are located upstream of the middle and late phage genes. Curiously, gp79, an inhibitor of host and early phage transcription by σ70-holoenzyme, activated transcription by the gp36-holoenzyme in vitro.
doi:10.1016/j.jmb.2012.01.002
PMCID: PMC3275717  PMID: 22261232
bacteriophage; genome; RNA polymerase; sigma factor; transcription regulation
2.  CRISPR transcript processing: a mechanism for generating a large number of small interfering RNAs 
Biology Direct  2012;7:24.
Background
CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated sequences) is a recently discovered prokaryotic defense system against foreign DNA, including viruses and plasmids. CRISPR cassette is transcribed as a continuous transcript (pre-crRNA), which is processed by Cas proteins into small RNA molecules (crRNAs) that are responsible for defense against invading viruses. Experiments in E. coli report that overexpression of cas genes generates a large number of crRNAs, from only few pre-crRNAs.
Results
We here develop a minimal model of CRISPR processing, which we parameterize based on available experimental data. From the model, we show that the system can generate a large amount of crRNAs, based on only a small decrease in the amount of pre-crRNAs. The relationship between the decrease of pre-crRNAs and the increase of crRNAs corresponds to strong linear amplification. Interestingly, this strong amplification crucially depends on fast non-specific degradation of pre-crRNA by an unidentified nuclease. We show that overexpression of cas genes above a certain level does not result in further increase of crRNA, but that this saturation can be relieved if the rate of CRISPR transcription is increased. We furthermore show that a small increase of CRISPR transcription rate can substantially decrease the extent of cas gene activation necessary to achieve a desired amount of crRNA.
Conclusions
The simple mathematical model developed here is able to explain existing experimental observations on CRISPR transcript processing in Escherichia coli. The model shows that a competition between specific pre-crRNA processing and non-specific degradation determines the steady-state levels of crRNA and is responsible for strong linear amplification of crRNAs when cas genes are overexpressed. The model further shows how disappearance of only a few pre-crRNA molecules normally present in the cell can lead to a large (two orders of magnitude) increase of crRNAs upon cas overexpression. A crucial ingredient of this large increase is fast non-specific degradation by an unspecified nuclease, which suggests that a yet unidentified nuclease(s) is a major control element of CRISPR response. Transcriptional regulation may be another important control mechanism, as it can either increase the amount of generated pre-crRNA, or alter the level of cas gene activity.
Reviewers
This article was reviewed by Mikhail Gelfand, Eugene Koonin and L Aravind.
doi:10.1186/1745-6150-7-24
PMCID: PMC3537551  PMID: 22849651
CRISPR/Cas; Transcript processing; Small RNA; CRISPR expression regulation; CRISPR/Cas response
3.  Redefining Escherichia coli σ70 Promoter Elements: −15 Motif as a Complement of the −10 Motif ▿ †  
Journal of Bacteriology  2011;193(22):6305-6314.
Classical elements of σ70 bacterial promoters include the −35 element (−35TTGACA−30), the −10 element (−12TATAAT−7), and the extended −10 element (−15TG−14). Although the −35 element, the extended −10 element, and the upstream-most base in the −10 element (−12T) interact with σ70 in double-stranded DNA (dsDNA) form, the downstream bases in the −10 motif (−11ATAAT−7) are responsible for σ70-single-stranded DNA (ssDNA) interactions. In order to directly reflect this correspondence, an extension of the extended −10 element to a so-called −15 element (−15TGnT−12) has been recently proposed. I investigated here the sequence specificity of the proposed −15 element and its relationship to other promoter elements. I found a previously undetected significant conservation of −13G and a high degeneracy at −15T. I therefore defined the −15 element as a degenerate motif, which, together with the conserved stretch of sequence between −15 and −12, allows treating this element analogously to −35 and −10 elements. Furthermore, the strength of the −15 element inversely correlates with the strengths of the −35 element and −10 element, whereas no such complementation between other promoter elements was found. Despite the direct involvement of −15 element in σ70-dsDNA interactions, I found a significantly stronger tendency of this element to complement weak −10 elements that are involved in σ70-ssDNA interactions. This finding is in contrast to the established view, according to which the −15 element provides a sufficient number of σ70-dsDNA interactions, and suggests that the main parameter determining a functional promoter is the overall promoter strength.
doi:10.1128/JB.05947-11
PMCID: PMC3209215  PMID: 21908667
4.  Transcription, Processing, and Function of CRISPR Cassettes in Escherichia coli 
Molecular microbiology  2010;77(6):1367-1379.
CRISPR/Cas, bacterial and archaeal systems of interference with foreign genetic elements such as viruses or plasmids, consist of DNA loci called CRISPR cassettes (a set of variable spacers regularly separated by palindromic repeats) and associated cas genes. When a CRISPR spacer sequence exactly matches a sequence in a viral genome, the cell can become resistant to the virus. The CRISPR/Cas systems function through small RNAs originating from longer CRISPR cassette transcripts. While laboratory strains of Escherichia coli contain a functional CRISPR/Cas system (as judged by appearance of phage resistance at conditions of artificial co-overexpression of Cas genes and a CRISPR cassette engineered to target a λ phage), no natural phage resistance due to CRISPR system function was observed in this best-studied organism and no E. coli CRISPR spacer matches sequences of well-studied E. coli phages. To better understand the apparently “silent” E. coli CRISPR/Cas system, we systematically characterized processed transcripts from CRISPR cassettes. Using an engineered strain with genomically located spacer matching phage λ we show that endogenous levels of CRISPR cassette and cas genes expression allow only weak protection against infection with the phage. However, derepression of the CRISPR/Cas system by disruption of the hns gene leads to high level of protection.
doi:10.1111/j.1365-2958.2010.07265.x
PMCID: PMC2939963  PMID: 20624226
5.  Temporal regulation of viral transcription during development of Thermus thermophilus bacteriophage φYS40 
Journal of molecular biology  2006;366(2):420-435.
SUMMARY
Regulation of gene expression of lytic bacteriophage φYS40 that infects thermophilic bacterium Thermus thermophilus was investigated and three temporal classes of phage genes -- early, middle, and late -- were revealed. φYS40 does not encode a DNA-dependent RNA polymerase (RNAP) and must rely on host RNAP for transcription of its genes. Bioinformatic analysis using a model of Thermus promoters predicted 43 putative σA-dependent −10/-35 class phage promoters. A randomly chosen subset of those promoters was shown to be functional in vivo and in vitro and to belong to the early temporal class. Macroarray analysis, primer extension, and bioinformatic predictions identified 36 viral middle and late promoters. These promoters have a single common consensus element, which resembles host σA RNAP holoenzyme −10 promoter consensus element sequence. The mechanism responsible for the temporal control of the three classes of promoters remains unknown, since host σA RNAP holoenzyme-purified from either infected or uninfected cells efficiently transcribed all φYS40 promoters in vitro. Interestingly, our data showed that during infection, there is a significant increase and decrease, respectively, of transcript amounts of host translation initiation factors IF2 and IF3. This finding, together with the fact that most middle and late φYS40 transcripts were found to be leaderless, suggests that the shift to late viral gene expression may also occur at the level of mRNA translation.
doi:10.1016/j.jmb.2006.11.050
PMCID: PMC1885378  PMID: 17187825
Thermus thermophilus; bacteriophage; bioinformatic promoter search; macroarray analysis; gene expression; leaderless mRNA
6.  Transcription regulation of the type II restriction-modification system AhdI 
Nucleic Acids Research  2008;36(5):1429-1442.
The Restriction-modification system AhdI contains two convergent transcription units, one with genes encoding methyltransferase subunits M and S and another with genes encoding the controller (C) protein and the restriction endonuclease (R). We show that AhdI transcription is controlled by two independent regulatory loops that are well-optimized to ensure successful establishment in a naïve bacterial host. Transcription from the strong MS promoter is attenuated by methylation of an AhdI site overlapping the -10 element of the promoter. Transcription from the weak CR promoter is regulated by the C protein interaction with two DNA-binding sites. The interaction with the promoter-distal high-affinity site activates transcription, while interaction with the weaker promoter-proximal site represses it. Because of high levels of cooperativity, both C protein-binding sites are always occupied in the absence of RNA polymerase, raising a question how activated transcription is achieved. We develop a mathematical model that is in quantitative agreement with the experiment and indicates that RNA polymerase outcompetes C protein from the promoter-proximal-binding site. Such an unusual mechanism leads to a very inefficient activation of the R gene transcription, which presumably helps control the level of the endonuclease in the cell.
doi:10.1093/nar/gkm1116
PMCID: PMC2275141  PMID: 18203750

Results 1-6 (6)