TRANSCRIPTIONAL REGULATORY PROTIENS

TRANSCRIPTIONAL REGULATORY PROTIENS

ASSINGMENT ON

REGULATION OF TRANSCRIPTION

Transcription is the process of transcribing DNA nucleotide sequence information into RNA sequence information. Both nucleic acid sequences usecomplementary language, and the information is simply transcribed, or copied, from one molecule to the other. DNA sequence is enzymatically copied by RNA polymerase to produce a complementary nucleotide RNA strand, called messenger RNA (mRNA), because it carries a genetic message from the DNA to the protein-synthesizing machinery of the cell.

Regulation of transcription is mediated by interaction of DNA sequences with the proteins. These are called as regulatory proteins.

Control of transcription is usually exerted at the step at which transcription is initiated.

The promoter region of a gene attracts the enzyme RNA polymerase and correctly orient it to begins its task of making an RNA copy of gene.

It is the combination of DNA sequences and the regulatory proteins molecules that acts as the switch to control transcription.

Diagram showing interaction of regulatory proteins with major grove of DNA

TRANSCRIPTIONAL REGULATORY PROTEINS

RNA polymerase bind to the DNA and initiate transcription at promoters, sites generally found near points at which RNA synthesis begins on the DNA template. The regulation of transcription initiation often entails changes in how RNA polymerase interacts with a promoter.

The basal rate of transcription initiation at the promoters of non house keeping genes is also determined by the promoter sequences, but expression of these genes is further modulated by regulatory proteins.

At least three types of proteins regulate transcription initiation by RNA Polymerase. These are –

1.Specific factors2.Repressors 3.Activators

1.Specific factors- It alters the specificity of RNA polymerase for a given promoter or set of promoter.

2.Repressors-It impede access of RNA polymerase to the promoter.

3. Activators- It enhances the RNA polymerase- promoter interaction.

1.SPECIFICITY FACTOR (REGULATORY PROTEIN)

In Prokaryotes- The sigma-subunit of the E-coli RNA polymerase holoenzyme Is a specificity factor. Most E-coli promoters are recognized by single sigma-Subunit (Mol wt 70,000). Under some condition for example bacterial stress sigma subunit is replaced by six other specificity factor like sigma32. It directs RNA polymerase to special set of promoters.

In Eukaryotes- Some of the general transcription factors, in particular the TATA-binding protein (TBP), may be considered specificity factors.

2.Repressors (REGULATORY PROTEIN)

In Prokaryotes- Repressor binds to specific sites on the DNA. In bacterial Cells such binding sites are called as Operators, are generally near a promoter

Regulation by means of a repressor protein that binds RNA polymerase to the DNA and blocks the transcription is called as Negative regulation. Repressor binding is regulated by molecular signals that is effector (binds to repressor causing conformational changes). Ex- tryptophan operon.

The interaction between effector or signal molecule and repressor either increases or decreases transcription.

In Eukaryotes- The binding sites for a repressor may be some distance from the promoter; binding has the same effect as in bacterial cells-inhibiting theassembly or activity of a transcriptional complex at the promoter.

ACTIVATOR (REGULATORY PROTEINS)

In Prokaryotes- Activators provide a molecular counterpoint to repressor; they bind to DNA and enhances the activity of RNA polymerase at a promoter; this is Positive regulation. Ex- Lac operon

Activators binding sites are often adjacent to promoter that are bound weakly or not at all by RNA polymerase alone, such that little transcription occurs in the absence of the activator.In Eukaryotes- Activator binds to the DNA sites , called enhancers, that are quite distant from the promoter, affecting the rate of transcription at a promoter that may be located thousands of base pair away. And some other activators are usually bond to DNA, enhancing transcription until dissociation of the activator is triggered by the binding of a signal molecule.

Diagram showing interaction of activator and repressor protein with DNA

Role of enhancer

SIGNIFICANCE OF THE REGULATION

Genes are functional unit of DNA.

Some genes for product that are required more or less all times in the cell for enzymes of central metabolic pathways of in more or less level. These genes are termed as house keeping genes.

Unvarying expression of genes are called constitutive expression.

For other gene product, cellular levels rise and fall in response to molecular signals, this is regulated gene expression.

This type of regulation is of two types-

1. Inducible- Process of increasing concentration called as induction.

2. Repressible- Process of decreasing concentration is called as repression.

For the differentiation of multicellular organism it is of most importance that a gene should express at a proper time in the form of a protein. This is the differentiation point in all the organism.

Transcriptional control is orchestrated by a large number of proteins, called transcription factors.

These factors can be divided into two major groups-1. General Transcription factors that bind at core promoter sites in associationWith RNA polymerase.

2.Sequence specific transcription factors that bind to various regulatory sites of particular genes. These second group of transcription factors act as either theTranscription activator that stimulate transcription or as Transcription Repressor that inhibit transcription.

TRANSCRIPTIONAL LEVEL CONTROL OR REGULATION BY TRANSCRIPTON FACTORS AND ITS INTERACTION WITH DNA

Studies making use of DNA microassays have also show much information about transcriptional level control or regulation.

MECHANISM OF TRANSCRIPTION FACTOR TO BIND TO DNA

The control of gene transcription is complex and is influenced by various circumstances , including the affinity of transcription factor for DNA sequencesAnd the ability of transcription factors bound at nearby sites on the DNA to interact directly with one another. This has been explored by some motif which Present in further section.

STRUCTURE OF TRNASCRITON FACTOR

Transcription factors contain at least two domain- 1.DNA-binding domain that binds to the a specific sequence of base pairs in the DNA. 2.Activation domain that regulates transcription by interaction with other Proteins and it contains a surface that promotes the binding of other proteins to make Dimers.

VARIOUS MOTIF TO EXPLAIN THE BINDING OF TANSCRIPTION FACTORS

There have been various motifs are given to explain DNA –transcription interaction and protein protein interaction which are as follows-

BINDING OF DNA WITH REGULATORY PROTEINS(TRANSCRIPTION FACTORS)

Helix turn helix motif- recognition helix, 20 amino acid resedues short alpha- helix segment.

Zinc finger motif- 30 amino acid residues with zinc ion, ex- frog Xenopus

Homeodomain- 60 amino acid residues ,called as homeodomain.

High mobility group proteins (HMG-box motif)

REGULATORY PROTEIN - PROTEIN INTERACTION

Basic helix-loop helix

Leucine zipper motif

Subunit mixing in eukaryotic regulatory proteins.

HELIX -TURN –HELIX MOTIF

DNA binding domain

of protein

In it there is a recognition helix, 20 amino acid residues short alpha- helix segment which play role in binding.

Entire Lac repressor

Surface having DNA binding domain

Separated protein and DNA sites to bind with

ZINC FINGER MOTIF

The zinc fingers of the

regulatory proteins

In a zinc finger about 30 amino acid residuesFrom an elongated loop held together at the base by a single zinc ion, which is coordinated tofour of the residues(four Cys, or two Cys and two His ). ex- frog Xenopus

Homeodomain is another type of DNA binding domain of 60 amino acid residues that binds with the regulatory proteins.

HOMEODOMAIN

Homeodomain

HIGH MOBOLITY GROUP PROTEINS (HMG- BOX MOTIF)

The name is given by the type of protein that is high mobility group protein.

The motif consist of three alpha helices organized such as that they have the capability of binding to DNA.

Transcription factors that posses HMG boxes are reffered to as “architectural”Factors; they activate transcription by bending the DNA, which is thought to protmote the interaction of other transcription factors bound nearby sites.

BASIC HELEX LOOP HELIX MOITF

Basic helix loop helix

Another common structureal motif occurs in some eukaryotic regulatory proteins implicated in the regulation at the time of the development of multicellular organisms.These proteins share aconserved region of about 50 amino acid Residues important in both DNA binding and protein dimerization

LEUCINE ZIPPER MOTIF

This motif is an amphipathic alpha helixWith a series of hydrophobic amino acidResidues concentrated on one side, with the hydrophobic surface forming the areaOf contact between the two polypetides of a dimer that is between to proteins Which are in dimer form.

Leucine zippers

Zipper region

SUBUNIT MIXING IN EUKARYOTIC REGULATORY PROTEINS

Several families of eukaryotic transcription factors or regulatory proteinsHave been defied based on close structural similarities. This similarity helps In binding if the proteins dimer is formed between indentical proteins then it is called ad Homodimer and if forms between different protein then called as heterodimer.

DNA SITES INVOLVED IN REGULATING TRANSCRIPTION

There are three sites which are present on the promoter which are involved In regulation of transcription. These are-

1.TAAT box2.CAAT box3.GC (rich regions)

On these sites regulatory protein or the transcription factors interact.

These has been detected by some techniques like Deletion mapping, DNA Footprinting and Genome- wide location analysis.

IN EUKARYOTES REGULATION BY SPECTFICTIY FACTOR -ROLE OF TATA BINDING PROTEIN AND TRANSCRITON FACTOR

Transcription factor also called a sequence-specific DNA binding factor, is a protein that binds to specific sequences of DNA and there by controls the transcription initiation.

As gene expression is controlled mainly at the level of transcription ,transcription factor play important role in gene expression.

RNA Polymerase II can not bind directly to eukaryotic promoter sites and initiate the transcription without the transcription factor. They need certain Regulatory proteins for the initiation of transcription.

The first regulatory protein to bind the assembly of preinitiation complex at TATA box of a typical polII promoter is the TATA binding protein. TheTranscription factor TFIID that binds directly to the TATA Box sequence.

TFIID consist of about 10 polypeptide subunits. Once initial binding to the DNA occurs, at least seven other transcription factors bind sequentially toTFIID, forming an extensive initiation complex, then bound by RNAPolymerase II.

TFIIA and TFIIB bind to the either side of TFIID , forming a site for the RNA polymerase binding.

TFIIF promotes RNA polymerase II binding to DNA binding complex.

TFIIE is known to stimulate the transcription.

TFIIH function is conjunction with TFIIE, both are required for the promoter clearance elongation, but initiation can occur without the TFIIE or TFIIH.

TFIIJ binds the RNA polymerase II , following the TFIIH and help in the start of transcription.

THREE DIMENSTIONALSTRUCTURE OF TBP

TRANSCRIPTION FACTORS

IN EUKARYOTES REGULATION BY ACTIVATOR AND REPRESSOR PROTEINS

Sucessful binding of RNA polymerase II holoenzyme at one of its promoter usually requires the action of other proteins.

1.Transcription activators-which binds to enhacer or upstream activator Sequences which are about 200 base pairs in length hving multiple sites for sequence specific transcription activators.

2.Chromatin modification and remodeling proteins-causes modification and remodeling of enzymes.

3.Coactivator proteins –required for regulation of transcription.These can be divides in to two groups-

1.Those which interact with basal transcription machinary2.Those that act on chromatin.

4.Basal transcription factors- all the transcription factors.

These factors make use o some enzymes that are as follows-1. Coactivators make use of HATs that is HISTONE ACETYLTRANSFERASES (having acetyl group addition activity)- which interact with histone protein and help in binding of regulatory protein or transcription factors with the DNA.HATS ARE ASSOCIATED WITH TRANSCRIPTIONAL ACTIVATION.

2.Chromatin modeling make use of HDACS that is HISTONE DEACETYLASES (having acetyl group removal activity)-which helps in remodelling of the chromatin. HDACS ARE ASSOCIATED WITH TRANSCRIPTION REPRESSION.Although much is not understood about trancription repression in eukaryots but it is considered that one of the key factor in doing this is DNA METHYLATION( it strongly correlate with gene expession).

Some enzymes complexes play role in chromatin modification in transcriptionexample of these are- Histone modification –GCN5-ADA2-ADA3(In yeast)SAGA/PCAF and Nua4(In eukaryotes) – having HAT( histone acetyl Transferase activity).

IN PROKAROTES REGULATION BY REPRESSOR PROTEINS-ROLE OF REPRESSOR IN LAC OPERON

In lac operon inducer is a molecule called allolactose, the galactosidase reaction produces small amount of lactose which is rearranged allolactose, beta(1,6) linkage.This molecule acts as an inducer for lac operon. Allolactose is a repressor protein that prevents it from binding to the operator.

Lac repressor protein binds to a DNA molecule, rapidly slides along DNA until reaches Operator and stops.

Portion of repressor fits into major groove of operator site DNA, shape of repressor suited to bind DNA.

RNA polymerase binds at promoter , located next to operator.

Repressor binds to move than one operator, bends the DNA segment containing promoter.

Bent promoter does not allow RNA polymerase binding and hence no initiation of transcription.

IN PROKAROTES REGULATION BY ACTIVATOR PROTEINS-

ROLE OF ACTIVATOR IN LAC OPERONOperons functioning only in the presence of controlling factors or proteins.

Lac operon function regulated by Catabolite Activator protein or cAMP receptor protein(CRP) and small cyclic nucleotide 3’,5’-cyclic adenosine monophasphte(CAP)

Promoter contains CAP site to which CAP protein binds before RNA polymerase attaches to promoter.

CAP is able to bind to CAP bends DNA about 90’ within 2 helical turns.

Interaction of CAP with RNA pol. Stimulates transcription.

STRUCTURE OF LAC REPRESSOR PROTIEN OPERATOR

The lac operon in the repressed state

Lac repressor bound to DNAConfermational change in

the lac repressor

IN PROKAROTES REGULATION BY REPRESSOR PROTEINS-ROLE OF REPRESSOR IN TRYPTOPHAN OPERON

When tryptophan is absent the trp repressor protein is in an inactive formCalled aporepressor.

The aporepressor can not bind to the operator of the trp operon.

Thus the operon is turned on in the absence of tryptophan and theTranscription occurs. RNA polymerase continues and transcribes trp operon genesTrp repressor decreases transcription.