Showing posts with label RNA Polymerase. Show all posts

Wednesday, February 19, 2014

Microbiology Daily Newsletter February 18, 2014 - Transcription Initiation

Microbiology Daily Newsletter

February 18, 2014 - Transcription Initiation

Hopefully you remember the basic idea of a gene and the mechanism of transcription as it applies to eukaryotic cells.

For bacterial transcription, we will first look at the promoter. You may recall the TATA box with eukaryotic promoters. In bacteria, we find the Pribnow-Schaller box, commonly just referred to as the Pribnow box. The Pribnow-Schaller box is a six nucleotide sequence TATAAT. QUESTION: Why six nucleotides?

With eukaryotic promoters, you may recall that the TATA box is not the only recognition sequence needed. This is also true of prokaryotic promoters. The Pribnow-Schaller box is found at -10 from the start of the gene. The complete promoter also contains a -35 recognition sequence, and is also comprised of six nucleotides. The -35 promoter element usually has a sequence of TTGACA, but note that this can vary among different bacterial taxa (usually at the class or family level taxa). NOTE: The -10/-35 promoter is used for normal house keeping genes. We will see some variation shortly.

The prokaryotic RNA Polymerase catalyzes the reaction of both coding and non-coding RNA, unlike eukaryotes that have job specific RNA polymerases (for example, you may remember that RNA-Pol I forms the 45S pre-rRNA, while RNA-Pol III forms tRNAs). The prokaryote RNA Polymerase Complex is a holoenzyme composed of RNA Polymerase and a Sigma (σ)factor. The eukaryotic RNA Polymerase complex has a specific RNA polymerase and a variety of initiation factors.

Below is an example of the bacterial transcription initiation complex. Note that you are seeing RNA Polymerase with 2 different σ factors, and that the different sigma factors have slightly different promoter recognition sites. When they bind, they move from a closed complex to an open complex; the open complex gaining its name from the opening of the DNA helix.

Add cFrom: Bush M , and Dixon R Microbiol. Mol. Biol. Rev. 2012;76:497-529. Initiation of transcription by the RNAP-σ70 (A) and RNAP-σ54 (B) holoenzymes. The σ70 factor directs the binding of polymerase to the consensus −10 (TATAAT) and −35 (TTGACA) sequences to form an energetically unfavorable closed complex (CC) that is readily converted into an open complex (OC) to initiate transcription. In contrast, the σ54 factor directs the binding of RNAP to conserved −12 (TGC) and −24 (GG) promoter elements that are part of the wider consensus sequence YTGGCACGrNNNTTGCW (where uppercase type indicates highly conserved residues, lowercase type indicates weakly conserved residues, N is nonconserved, Y is pyrimidines, R is purines, and W is A or T) (10). This forms an energetically favorable CC that rarely isomerizes into the OC. In order to form the transcription “bubble,” a specialized activator (a bacterial enhancer binding protein [bEBP]) must bind and use the energy from ATP hydrolysis to remodel the holoenzyme. aption

Once the RNA Polymerase-σ factor Holoenzyme is bound in the open complex form, transcription can proceed to the elongation phase. The next issue specific for prokaryotic transcription will be termination.

Daily Challenge

Sigma factors play a critical role in coordinating bacterial cell physiological states. Below is a list of common sigma factors.

Sigma Factor	Gene	Function
s⁷⁰	RpoD	Primary s factor, Housekeeping
s¹⁹	FecI	Regulates fec gene for iron transport
s²⁴	RpoE	Extreme heat stress
s²⁸	RpoF	Flagellar genes
s³²	RpoH	Heat shock
s³⁸	RpoS	Starvation/Stationary phase

Along with these can be found anti-sigma factors that block the function of expressed sigma factors (a form of regulation). Discuss the role of sigma factors as means of global gene regulation. Why is having a sigma factor system beneficial to bacteria? How does this differ from eukaryotic promoters?

Tuesday, September 10, 2013

Daily Newsletter - September 10, 2013 - Transcription

Daily Newsletter

September 10, 2013 Transcription

Transcription is the genetic process where a single strand of DNA acts as a template for the construction of a complementary RNA strand. Generally when talking about transcription, we will be talking about the formation of messenger RNA (mRNA), which carries the code for one gene to a ribosome where it is translated into a protein.
DNA holds the "permanent" copy of the genes needed to make a functional organism (nothing is really permenant). Think of DNA as a locked safe where you hold all your company's blueprints, patents and documented procedures. You don't want to loose these, or risk that they might be changed. You only bring them out to make copies of them, then they go back to the safe. This is what happens with your DNA. You keep it tightly locked up (in a double-helix that is coiled around histones, and then possibly supercoiled), and open it up only when you NEED to make a copy. Notice how NEED is highlighted? Do you think it might be an important concept?

In eukaryotic DNA every gene starts with a promoter. This is a sight of ~8 nucleotides visible in the major groove of DNA. The transcription complex recognizes this sequence as a "START" indicator. The main core of the transcription complex will be RNA polymerase. This enzyme works to build a strand of RNA complementary to DNA. The name polymerase indicates that it is involved with dehydration synthesis polymerazation reactions (taking one nucleotide, and adding it to a growing chain of nucleotides). Like DNA polymerase, RNA polymerase builds in the 5' to 3', and builds phosphodiester linkages between nucleotides.

But RNA polymerase can not act alone. In eukaryotic systems, initiation factors are needed to recognize the promoter region, and then to correctly align the RNA polymerase. To the left is a great picture showing the initiation complex and the RNA polymerase II holozyme (RNA polymerase II with all associated protein structures). You are not responsible for knowing all of the factors needed to initiate eukaryotic transcription, but you do need to start understanding the concept that it takes multiple factors to identify a promoter and start RNA polymerase. What do you think you need in order to recognize a specific sequence of nuclotides?
As you can see, TATA Binding Protein (TBP) is the first structure to attach to DNA. It recognizes the TATA sequence in the major groove of the DNA double helix. It then forces the the DNA to bend, and acts as a signal to other enzymes directing interactions with DNA. A cascade of reactions occur to then produce the Preinitiation Complex, which ensures that the transcription complex is positioned correctly over the Transcription Start Site, and begins the unwinding (sometimes referred to as denaturation) of the double helix. The Transcription Complex then begins to read the template strand of DNA, and makes an RNA copy (Elongation). [NOTE: Bacteria use proteins known as sigma factors to help find promoter regions and initiate transcription. There are different sigma factors linked to different environmental and physiological states, such as the Heat Shock Sigma factor which alter's the bacteria's ability to deal with higher temperatures)]
Elongation works due to base complementarity. Ribonucleotide triphophates are brought into the transcription complex, and are added to the free 3' end of the growing RNA strand. During the elongation phase, the RNA polymerase continues to add nucleotides to the growing RNA strand.
At some point, the RNA polymerase comes to a termination sequence. We are not going to spend a lot of time on termination (you are not held responsible for the various models). There are a couple different models of eukaryotic transcription termination. The main feature is that there is a signal sequence of deoxyribonucleotides in DNA that signals the end of transcription. Once this signal sequence is found, RNA polymerase is removed and the new transcript (new RNA molecule) is released.
mRNA processing: Once transcription is complete, in eukaryotes, the RNA needs to be processed. The following is a quick reference for mRNA processing:

5' capping: To protect the mRNA from ribonucleases (RNA degrading enzymes) that attack the 5' end, 7-methylguanosine is added to the 5' end. Usually, the 5' ribonucleotide is replace by this compound. Additionally, methyl groups can be added to the sugar-phosphate backbone to further protect the mRNA.
Polyadenylation: In maturing RNA to mRNA, a poly-A tail is added (usually after cleaving off a small section of the 3' end). This process adds ~250 adenyls to the 3'end of the molecule. This is needed to stabilize the molecule and facilitate export through the nuclear pores. As mRNA is translated, the poly-A tail gets shorter. When short enough, the mRNA is degraded. Thus, the polyadenylation (poly-A tail) is responsible for setting a time limit to the mRNA.
Splicing: The RNA is composed of both coding (exon) and non-coding (intron) regions. To mature into mRNA, the introns have to be removed, and the remaining exon spliced together. This job is the responsibility of the splicosomes.

The above image is a quick reference to the effects of splicing.

The above image is a quick reference to the effects of the splicosome.

Once RNA has been processed (matured), it is ready to be used in translation (protein synthesis). NOTE: Bacterial RNA does not undergo processing. The bacterial RNA transcript is immediately translated.

Daily Challenge

Transcription In your own words, discuss the process of transcription, and the formation (maturation) of mRNA. Remember that we have focused on eukaryotic transcription. Briefly, how does prokaryotic (specifically bacterial) transcription differ from eukaryotic transcription?
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