Last modified on 11 December 2013, at 01:50

Structural Biochemistry/Transcription

Basics of TranscriptionEdit

Traewf Transcription is similar to DNA replication in that DNA is used as a template to make a new nucleotide strand (RNA). The newly synthesized RNA strands are complementary to the DNA template strand.

RNA polymerase uses ribonuceloside triphosphate (rNTP) to synthesize mRNA strands (rATP, rUTP, rCTP, and rGTP) in the 5'->3' direction.

Transcription can be broken down into 3 steps:

1. Initiation. Transcription begins when RNA polymerase binds to a DNA region known as a promoter. Additional transcription factors are required to hold the RNA polymerase to the correct region of the DNA. After RNA polymerase binds to the promoter region, it melts 10-15 nucleotide base pairs around the transcription start site, allowing for rNTPs to bind to the template strand. Initiation ends when the first rNTP is linked to RNA polymerase by a phosphodiester bond. (Unlike DNA replication, no primer is needed)

2. Elongation: RNA polymerase leaves the start site and travels down the template in the 3'->5' direction. The DNA helix opens ahead of RNA polymerase during this process due to helicase.

3. Termination: Rna polymerase releases from the DNA template strand and leaves DNA.

Prokaryotic Transcription: OperonsEdit

Transcription is very similar in both prokaryotes and eukaryotes in that there is an intiation step, elongation step, and termination step. However, they also have their differences.

Prokaryotes contain operons while eukaryotes do not. Operons are clusters of related genes involved in a similar function and are often found in a contiguous array. Operons are controlled by a single promoter, and as a result, transcription produces 1 mRNA that can be translated into multiple proteins. If there were no operons, there would have to be separate promoters for each gene. Operons help make transcription more efficient; for example:

With Operon:

  |Promoter|Gene A|Gene B|Gene C|  ---transcription---> mRNA ---translation---> protein A + protein B + protein C

Without Operon:

  |Promoter|Gene A| ---transcription---> mRNA A ---translation---> protein A
  |Promoter|Gene B| ---transcription---> mRNA B ---translation---> protein B
  |Promoter|Gene C| ---transcription---> mRNA C ---translation---> protein C

While an operon provides the advantage of being able to initiate transcription at one point and transcribe many genes, it has its disadvantages as well. One disadvantage is that if the promoter for the operon sequence is mutated, all the genes in the operon cannot be transcribed.

An operon consists of 3 parts:

1. Structural Genes
2. Promoter region
3. Operator region

The structural gene encodes for proteins. All structural genes will turn into a single mRNA that encodes for multiple proteins.

The promoter region is where RNA polymerase binds to DNA to initiate transcription. Not all promoters will have the same sequence. Strong promoters will have a similar nucleotide sequence as a known consensus sequence. Weak promoters will have a different sequence.

The operator region is located next to, and overlaps with the promoter region. It is the site where a repressor can bind. When a represson binds to an operator, RNA polymerase will not be able to bind to the promoter, and as a result transcription will not occur.

Inducible Operons

An inducible operon is an operon where a substance is required to be bound before transcription will occur, it is normally "off" but when a substance binds it is turned "on." The lac operon is an example of an inducible operon. It encodes 3 enzymes involved in the metabolism of lactose. The lac operon has 4 regions:

1. CAP binding site: important to increase the rate of transcription of an operon
2. Promoter: location where RNA polymerase binds
3. Operator: location where a repressor binds
4. Genes (ZYA): 
  Gene Z encodes for Beta-galactosidase, which breaks down lactose
  Gene Y encodes for galactosidase primase, a transporter protein that allows lactose to get into the cell
  Gene A encodes for galactosidase transacetylase

Bacteria prefer to metabolize glucose over lactose. Given this fact, we can see three different scenarios:

1. No Lactose, High Glucose

If there is no lactose, the lac operon will not be turned on. A repressor will bind to the operator which overlaps the transcription start site, preventing RNA polymerase from binding at the promoter, and thus preventing transcription of the lac operon. This is important for bacteria to save energy. Because they prefer to metabolize glucose, there is no need to turn on the lac operon in the presence of no lactose and high glucose.

2. High Lactose, High Glucose

Lactose will bind to the repressor, causing a conformational change that forces the repressor to unbind from the operator. As a result, RNA polymerase can bind to the promoter and allows for transcription to occur, however, it is a weak transcription.

3. High Lactose, Low Glucose

Lactose will still bind to the repressor and force it to unbind from the operator (due to the high amount of lactose present). However, the low glucose levels will cause an increase in cyclic AMP. The increase in cAMP will bind to the CAP binding site. This then increases RNA polymerase's binding to the promoter, resulting in high amounts of transcription.

Repressible Operon

A repressible operon is an operon that where transcription is normally "on," but when a substance binds it turns "off." It is the opposite of an inducible operon.

The Trp Operon is an example of a repressible operon, and is involved in the synthesis of the essential amino acid tryptophan. Tryptophan is either made, or obtained from the environment. The Trp Operon is normally on, transcribing the RNA needed to synthesize tryptophan, however, in the presence of tryptophan (obtained from the environment), the operon is turned off in order to save energy.

Prokaryotic Transcription: Transcription Factors (Sigma)Edit

The function of a transcription factor is to bring RNA polymerase and the promoter together. It will bind to RNA polymerase and at the same time, associate with the DNA promoter. Transcription factors exist in both prokaryotes and eukaryotes.

An example of a prokaryotic transcription factor is the "Sigma Factor." The sigma factor is involved in locating RNA polymerase to the correct location.

Eukaryotic Transcription: RNA PolymeraseEdit

There are three different types of RNA Polymerase in eukaryotes:

1. RNA polymerase I: makes rRNA
2. RNA polymerase II: makes mRNA, miRNA, and splicing RNA
3. RNA polymerase III: makes tRNA, rRNA, and splicing RNA

The structures of all three RNA polymerases are very similar and highly conserved. The components include a site for rNTP to enter, and a site for phosphodiester bond formation. RNA polymerase II contains an area known as the carboxy terminal domain (CTD), which is RNA pol II specific. CTD is a string of seven amino acid repeats, which is found to repeat 52 times in vertebrates. It is essential for viability, and RNA polymerase II cannot function without it. Before transcription, CTD is non-phosphorylated, and after the initiation step of transcription, CTD becomes phosphorylated.

Eukaryotic Transcription: Promoter, Proximal Promoters, and EnhancersEdit

The promoter tells the cell where to start transcription. Transcription factors identify and bind to promoter regions, and also help to recruit RNA polymerases.

Proximal promoters and enhancers are both sites for transcription factors to bind as well. Proximal promoters are about 200 base pairs upstream of the start site. Enhancers are further away from the start site, and can be found up to 50,000 base pairs up or downstream of the site.

Promoter regions were identified through two different experiments: 5' deletion analysis and linker scanning analysis.

Prokaryotic vs. Eukaryotic TranscriptionEdit

A few differences between Eukaryotic and Prokaryotic transcription include: Eukaryotes have multiple general transcription factors, lack operons, and have a genome packed into chromatin. Prokaryotes on the other hand, have one general transcription factor, have operons, and have a genome located in plasmids.

Looking at and comparing the three steps of transcription between Prokaryotes and Eukaryotes:

1. Initiation: the binding of RNA polymerase to double-stranded DNA.

For prokaryotes: RNA polymerase, in its complexity, contains a core that has to bind to the promoter region of the DNA template. Another subunit, known as sigma, is what makes this possible by finding and binding to the promoter region using lots of weak H-bonds with the base pairs (with the culmination of many weak H-bonding, a strong net force is seen). So, the RNA polymerase simply slides along the DNA, and it either finds a promoter, or dissolves and starts somewhere else. If it finds a promoter, it proceeds to unwind the DNA, which leads to elongation. Prokaryotes have two promotors sites located upstream of the first nucleotide to be transcribed. They are the Pribnow box, which is located 10 nucleotides upstream and has the sequence TATAAT and the -35 region which has the sequence TTGACA.

For Eukaryotes: Eukaryotes are more complicated in their initiation phase. Firstly, the RNA polymerase doesn't randomly scale a DNA for promoter regions. Rather, transcription factors are used to create specific instances in the promoter regions for the RNA polymerase to bind to. In eukaryotic cells, there are also 3 different kinds of RNA polymerase, each that transcribes a different type of RNA. Once a RNA polymerase binds to its respective promoter region (equipped with transcription factors), it creates a transcription initiation complex, which traverses the DNA. Eukaryotes also have two promotor sites, one is called the TATA or Hogness box which is at location -25 and has the sequence TATAAA, and the CAAT box, which is located at -75 and has the sequence GGNCAATCT. Transcription is initated by stimulation by the enhancer sequence.

2. Elongation: the covalent addition of nucleotides to the 3' end of the growing polynucleotide chain.

For prokaryotes: As the DNA is unwound, its base pairs are now available for binding. The first ribonucleoside triphospate (RNA building blocks) binds to it. The RNA polymerase loses its sigma subunit, leaving only the core. The unwound DNA sites provide sites for the RNA building blocks to H-bond to (in their correct base pair). Also, the triphosphates are used like train links, that covalently form phosphodiester bonds with each new RNA block.

For Eukaryotes: As the complex moves across the DNA, it unzips it and allows for Watson-Crick base-pairing to occur with transient RNA building blocks, linking them together using phosphodiester bonds.

3. Termination: the recognition of the transcription termination sequence and the release of RNA polymerase.

For prokaryotes: Elongation continues until it reaches a stop signal found on the DNA. The RNA polymerase core dissolves, and the DNA rewinds. The termination signal in E. Coli is a base-paired hairpin which is rich in guanine and cytosine. These two nucleotides binds complementary to one another creating a hair-pin turn which is then followed by several uracil nucleotides. That hairpin acts like a knot to the RNA strand that is being made, so the RNA detaches from the DNA template. The polymerase leaves shortly after, and the DNA is rewound. Transcription can also be stopped by a rho protein which causes the mRNA to fall of the template DNA strand.

For Eukaryotes: When the RNA complex reaches a termination signal on the DNA, the RNA polymerase is simply detached from the DNA allowing it to rewind. The resulting RNA is then processed. The newly-transcribed mRNA is future processed by adding a cap to the 5' end and a poly(A) tail to the 3' in a process called polyadenylation. It adds several adenine residues to the 3' end of the mRNA. The cap and poly(A) tail act to stabilize the mRNA molecule and prevent it from degrading.

Transcription


Chromatin ModificationEdit

DNA and proteins make up complex chromatin, and is can be found as either enchromatin or heterochromatin. Enchromatin is loosely condensed while heterochromatin is tightly condensed. Chromatin condensation is important because it determines transcription activity: if chromatin is too tightly condensed (heterochromatin), then the transcription factors and RNA polymerase are not able to get in; however, if chromatin is loosely condensed like in enchromatin, then transcription factors and RNA polymerase can more easily access the chromatin and start transcription. This means that genes containing enchromatin are highly likely to be transcribed, whereas heterochromatin genes are less likely to be transcribed.

30nm Chromatin Structures

Chromatin compaction and relaxation is regulated by modifying histone tails. When histone is attached to the acetyl group, due to the neutralization of the positive charge on the histone, the interaction between the negative charge on the DNA and the positive charge on the histone becomes weaker. As a result, RNA polymerase can easily access to the DNA, and thus, this process facilitates the transcriptional activity in vivo. In contrast, when histone is deacetylated, meaning that acetyl group is removed from the histone tail, the chromatin structure becomes more compacted, and accordingly, transcription is repressed.


http://www.broadinstitute.org/chembio/lab_schreiber/anims/animations/smdbHDAC.php