Thursday, May 6, 2010

Bacterial Transcription: Initiation, Polymerization, Termination

After the last post, I thought I would be updating more often, but that just didn't happen.  Either way, here's a post about bacterial transcription!
Bacteria as an Experimental System

Bacteria are a common genetic system, but why exactly do we use these tiny organisms to perform so many experiments?  Because they’re easy to use, of course.  There area  number of benefits to using bacteria, including:
Establishing basic biological principles
Genetic manipulation
Short generation time
Simple growth conditions
-  High population density 
- Ability to witness rare events
Ability to select for rare variants

Bacterial Transcription
Transcription is function that has been heavily studied in bacteria.  This first step in gene expression is facilitated by a single RNA polymerase of six subunits.  Eukaryotes, in contrast, have four polymerases (I, II, and III, as well as a mitochondrial or chloroplast polymerase).  In bacteria and eukaryotes, the initiation of transcription requires a complex of proteins to assemble and facilitate polymerization of RNA from DNA templates.  

Bacterial RNA polymerase consists of six subunits, as mentioned previously.  The β and β’ subunits perform the polymerization reaction.  Two α subunits regulate the frequency of initiation.  The ω subunit is involved in stability and assembly of the polymerase enzyme. 

RNA polymerase first binds to the promoter region of DNA using a σ factor, which binds two specific regions of the promoter. The core polymerase and σ factor slide along DNA until they come upon a promoter.  This closed complex that finds the promoter converts to an open complex (not requiring any ATP for this action), which favors the separation of the DNA strands.  At this point, RNA polymerase begins to make short RNA segments, as it “stutters” along the DNA.  Small RNA oligos are formed, and σ factor begins to dissociate from the polymerase enzyme.  At this point, elongation of RNA transcripts can occur, which results from a tightening of the clamp and the formation of the RNA exit channel.  During elongation, RNA polymerase adds nucleotides to the growing RNA transcript at a rate of about 50 per second.  With σ factor dissociated, the “rudder” of RNA polymerase pries the DNA/RNA hybrid apart. 

When RNA polymerase is to stop the transcription of a gene, it has a few options.  First, the gene itself may have an AT-rich region that forms secondary structures that inhibit transcription after they have been copied.  These hairpin secondary structures may open the exit channel, and due to the less stable A-U base-pairing between the DNA and RNA, the transcript is released.  Additionally, there is a rho-dependent transcriptional termination method.  Rho is a hexameric protein that wraps approximately 60 bp of mRNA.  Rho, once bound to mRNA, activates and uses its ATPase activity to move as an RNA-DNA helicase.  Once it has become active, rho begins to unwind the RNA from the DNA, and when it approaches the active site of RNA polymerase, the transcript is released from the DNA. 

Bacterial genes in general can be found  in either direction on the genome and are very rarely overlapping.  RNA polymerase recognizes a distinct region on the chromosome to initiate transcription.  To identify this site, DNA footprinting is used.

DNA footprinting:
1.      Bind RNA polymerase to a DNA strand of known length
2.      Randomly cleave the DNA by nuclease or chemical agents
3.      Remove RNA polymerase from the DNA
4.      Separate the DNA strands on an agarose gel.

By DNA footprinting, it was recognized that there is a specific region that is “empty” (the footprint) on the agarose gel corresponding to where RNA polymerase binds.  Genetic analysis has identified two regions where RNA polymerase binds: at -35 and -10 relative to the initiation site.  The consensus sequences are TTGACA and TATAAT, respectively.  

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