Bacterial transcription

Figure 37-6. The predominant bacterial transcription termination signal contains an inverted, hyphenated repeat (the two boxed areas) followed by a stretch of AT base pairs (top figure). The inverted repeat, when transcribed into RNA, can generate the secondary structure in the RNA transcript shown at the bottom of the figure. Formation of this RNA hairpin causes RNA polymerase to pause and subsequently the p termination factor interacts with the paused polymerase and somehow induces chain termination.

A bacterial two-hybrid system has been developed that, similar to the yeast system, functions via activation of transcription (Dove and Hochschild, 1998 Joung et al., 2000). RNA polymerase (RNAP) in E. coli consists of an enzymatic core composed of the a, (3, and (3 subunits in the stoichiometry a2(3(3, and one of several c factors that enable the enzyme to recognize specific promoters (Heilman and Chamberlin, 1988). Many bacterial transcriptional activator proteins bind the promoters they regulate and interact directly with subunits of RNAP. The most commonly observed contact is between activator proteins and the a subunit of RNAP (Ebright and Busby, 1995). The function of the a subunit is to initiate the assembly of RNAP by forming a dimer (Igarashi et al., 1991). 

Because of the preceding properties, our profile procedure appears to produce highly sensitive and specific common pattern representations from limited numbers of defining sequences compared with other current methods (Figs. 5 and 7). This was shown by the construction of such profiles from more than 50 completely unrelated functional families. In more than 90% of the families, the sensitivity and specificity are more than 98%. This is also supported by the repeated sampling study of the complex bacterial transcription initiation factors. Finally, these methods allow for the localized recognition of entire domains within multidomain structures, as seen in Fig. 6. 

The absence of a nuclear membrane is a characteristic of bacteria that has a profound effect on transcription. Bacterial transcripts are processed rapidly, and their 5 ends often enter ribosomes and are directing protein synthesis, while the 3 ends of the genes are still being transcribed. In contrast, most eukaryotic RNA transcripts must be processed and transported out of the nucleus before they can function. As consequence, many aspects of the control of transcription differ between prokaryotes and eukaryotes. 

Bacterial RNA polymerase, the target for the rifamycin class of antibacterial agents, is the enzyme responsible for transcription of genomic DNA in bacteria [40], Like DNA gyrase, RNA polymerase is a multifunctional, multisubunit enzyme with multiple active conformations. This increases the number of possible mechanisms of inhibition of RNA polymerase. For example, in addition to the 3 subunit, which is the apparent target for rifamycin, bacterial transcription initiation is a unique process in which the o subunit plays a unique role in the recognition of bacterial promoter sequences [41], Alternative c subunits, such... 

Bailey, M.J.A., Hughes, C., Koronakis, V. RfaH and the ops element, components of a novel system controlling bacterial transcription elongation. Mol Microbiol 26 (1997) 845-851. 

On the other hand, another attractive idea for the role of secondary metabolites has been propounded by Davies and coworkers.74 They demonstrated that some antibiotics at low concentrations alter global bacterial transcription patterns.74 The precedent discovery was done by Murakami et all5 on the binary activity of... 

This regulatory arrangement is most simply illustrated by bacterial transcription in which the structural gene is prefaced by a regulatory region including promoters that bind regulatory proteins ... 

The regulation of bacterial transcription is well illustrated by the lactose operon (lac operon) of the colon bacterium Escherichia coli in which the upstream region successively (from the 5 end of the sense strand) includes a promoter (P ) for the gene (I) coding for a repressor protein (the lac repressor), a CRP binding site , the promoter for the lac operon (P), and finally an operator site (O) that prefaces the Z, Y and A structural genes of the operon ... 

From the biochemical point of view, the unique mode of action of some of the ansamycins, in particular of rifampicin, has aroused much interest. Rifampicin and several other ansamycins have been shown to inhibit bacterial transcription very specifically and at extremely low concentrations by interacting exclusively with DNA-dependent RNA polymerase. This unique action has spurred many investigations on the effects of ansamycins in a variety of viral and eukaryotic systems. More recently maytansine and related compounds have been found to be very potent antimitotic agents and to have interesting antitumour activities. 

More recently Michnick and co-workers have introduced a dihydrofolate reductase complementation system, which seems to be particularly robust [61 - 65], They attribute the success of this system to the fact that the N-terminal (1 - 105) and C-terminal (106 - 186) DHFR fragments do not fold until they are dimerized. In addition to the obvious selection for essential metabolites dependent on the reduction of dihydrofolate to tetrahydrofolate, protein-protein interactions are detected based on the retention of a fluorescein-methotrexate conjugate. Several other enzymes have been employed for the design of complementation assays, including green fluorescent protein, which allows screens based on fluorescence or FRET [66 - 68]. As with the bacterial transcription assays, these complementation systems are new. It will be interesting to see if, as the selections are optimized, these systems prove competitive with the Y2H assay. 

Bacteria do not contain nuclei, so transcription and translation occur simultaneously. A single RNA polymerase produces mRNA, rRNA, and tRNA in bacteria. Bacterial transcripts (e.g., those from E. coli) do not contain introns. 

Smith, A.J. and Savery, N.J. (2008) Effects of the bacterial transcription-repair coupling factor during transcription of DNA containing non-bulky lesions. DNA Repair, 7, 1670-1679. 

Muller-Hill, B. 1998. Some repressors of bacterial transcription. Cure Opin. Microbiol 1 145-151. 

Although out of the scope of this review, it is notable that similar trends can be clearly seen in various receptors, where the acquisition of specificity for a new effector exploits the promiscuity of existing recep-tors. " As also demonstrated with bacterial transcription factors, new effector specificities can then be acquired by natural evolution, or laboratory rounds of mutagenesis and selection, often with weak negative trade-offs with respect to the original effector. ... 

Protoporphyrin is a fluorescent compound whereas heme is not, and therefore we screened a population of Tn5-induced mutants that formed fluorescent colonies imder ultraviolet light. The resultant mutant, strain LODTM5, had the desired phenotype in that it accumulated protoporphyrin only under iron limitation, it was not defective in the last two steps of the heme pathway, and it was not a heme auxotroph. Strain LODTM5 carries a loss-of-function mutation in a new gene named irr (iron responsive regulator) that encodes a protein predicted to contain a helix-tum-helix motif of the GntR family of bacterial transcriptional regulators. 

Adel M. Talaat, Preston Hunter and Stephen Albert Johnston, Genome-directed primers for selective labeling of bacterial transcripts for DNA microarray analysis. Nature Biotechnology, 18 (2000), 679-682. 

A great deal of interest has been shown in the study of DNA-protein interactions [30]. As such, many reviews cite the application of AFM to these interactions [30,104,112,113]. AFM has been used to provide structmal information on DNA binding sites and the stoichiometry of proteins that bind to the DNA [62]. The regulation of bacterial transcription [104], and the mechanisms involved in its initialisation are of considerable interest [114,115], as is the application of AFM in order to elucidate mechanisms of DNA-protein interactions that are involved in the repair [62] and replication [90,116] of DNA. 

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