Saturday, May 8, 2010

Bacterial Transcription: Induction and The Lac Operon

A continuation from previous transcription posts: Regulation and Attenuation and Initiation, Elongation, and Termination.

Gene induction is a phenomenon that is incredibly fast, taking only two to three minutes for a cellular response.  During induction, the actual enzyme levels in the cell rise, and inhibitors of protein synthesis prevent induction (providing further evidence that it is, in fact, protein synthesis that is necessary for induction).  The lac operon is the most commonly studied gene that uses induction for regulation.

During initial studies of the lac operon, there were two types of genes considered: structural and regulatory genes.  Structural genes are those that encode the actual metabolic enzymes; regulatory genes are involved in controlling the expression of the structural genes.  The lac operon was convenient for study because it had an observable phenotype (the production of a gene in the presence of glucose or lactose) and because mutants could be generated that had different phenotypes.  After mutagenizing bacteria, the researchers screened E.coli on plates with glucose and X-galactose.  Colonies of bacteria that were inducible turned white and did not express β-galactosidase, and regulatory gene mutants would be able to express β-galactosidase in the absence of lactose (and turn blue).  Mapping of the genes that were responsible for these phenotypes led to the identification of the o and i regions.  Mutations in either of these regions resulted in a constitutive phenotype. 

Further analysis of the lac operon and induction led to the creation of a model:  the i region codes for the inducer, which binds the lac operon DNA in the promoter region (identified via DNA-binding assays and footprint analysis).  Later structural studies identified the i protein contains an HTH motif, as well as IPTG-binding domains.  The i gene, called the repressor can bind the promoter region of the lac operon and prevent RNA polymerase from binding.  With the presence of glucose but not lactose, the lac repressor binds the operator sequence of the genome and it prevents RNA polymerase and its helper protein CAP (bound to cAMP).  CAP-cAMP induces a bend in the DNA, which allows RNA polymerase to bind, and CAP has its own binding site the DNA that helps to position the polymerase.  With the presence of lactose, the repressor binds to the lactose and no longer binds the operator.  Therefore, RNA polymerase can bind the promoter sequence and promote transcription of the lac genes.  However, in the absence of glucose, which is indicative of a high concentration of cAMP (low ATP), CAP binds cAMP and promotes stronger interaction of polymerase and the lac promoter.   

Friday, May 7, 2010

Bacterial Transcription: Control and Attenuation

A continuation from yesterday's post: Bacterial Transcription Initiation

Transcriptional Control and Attenuation
Like eukaryotes, bacteria must be able to control their gene activity.  Gene expression can be controlled at the transcriptional level in a few ways.  One is via alternative sigma factors (the protein that binds the -10 and -35 sites and positions RNA polymerase), which are involved in controlling expression of specialized operons.  For example, σ32 is involved in regulating heat shock genes, σ28 is for genes involved with motility and chemotaxis, σ54 is involved in nitrogen metabolism, and σ70 helps transcription of most genes. 

Another method of transcriptional control is via attenuation.  The most frequently cited example of attenuation is the trp operon, which has been studied extensively.  Initial observations indicated that when tryptophan was present for the bacteria, mRNA corresponding to the trp gene were short.  However, when tryptophan was limiting in the media, the mRNA transcript was longer.  If the researchers removed a short sequence of DNA, the mRNA was transcribed in full and genes were fully expressed.  This short sequence of DNA was termed the attenuator, or premature transcriptional stop. 

The trp operon codes for an mRNA with four different regions that can differentially bind to each other:  The second region can bind the first or third; the third can bind the second or fourth.  The first region contains two successive codons for tryptophan incorporation, which important for determining how the transcript is formed.  With high tryptophan, the ribosome moves along through the first region, without stopping at the successive tryptophan codons.  Because RNA polymerase has not had time to release the transcript before the ribosome translates through region one, causing regions three and four to bind, polymerase is forced off the mRNA and transcription is prematurely stopped.  This results in a shortened transcript when the cell has sufficient tryptophan.  In contrast, with low tryptophan levels in the cell, the ribosome will stall at the successive tryptophan codons because it is not able to quickly translate the mRNA.  This stalling allows for RNA polymerase to continue on its merry way and finish the full transcript because regions two and three (not three and four) bind.  

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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