Gene Regulation Expression in Eukaryotes & Prokaryotes

SDKMarch 30, 2013

1

Gene Regulation in Prokaryotes and Eukaryotes

Gene is the sequence of nucleotides in DNA that code one mRNA molecule or one polypeptide chain

In prokaryotes the primary control point is the process of transcription initiation

In eukaryotes expression of gene into proteins can be controlled at various locations.

2

Check Points for Gene Expression in Eukaryotes

1. Synthesis of proteins is controlled right from the chromatin stage.

2. Expression of gene is controlled at many steps during the process of transcription and translation.

3

1.Chromatin Structure

Two forms of chromatin Euchromatin – A lesser coiled transcriptionally

active region which can be easily accessed by the RNA polymerases.

Heterochromatin – A highly condensed transcriptionally inactive region. The genes in this region cannot be accessed by the RNA polymerases for active transcription.

4

1.Chromatin Structure

Mechanisms which affect the chromatin structure and hence the expression of gene are:

1.Histone modifications – These modifications make a region of gene either transcriptionally active or inactive.

a)Acetylation(addition of an acetyl (CH3CO) group to one of the

histone) • ↑Acetylation ----↓ Condensation of DNA ----- ↑

Transcription of genes in that region

5

1.Chromatin Structureb. Methylation

• Methylation of histone H4 on R4 (arginine residue at the 4th position) →→ opens the chromatin structure →→ leading to transcriptional activation

• Methylation of histone H3 on K4 and K79 (lysines residues at the 4th and 79th position) →→ opens the chromatin structure →→ leading to transcriptional activation

• Methylation of histone H3 on K9 and K27 (lysines residues at the 9th and 27th position) →→ condenses the chromatin structure →→ leading to transcriptional inactivation

6

1.Chromatin Structure

c) UbiquitinationUbiquitination of H2A – Transcriptional inactivationUbiquitination of H2B - Transcriptional activation

2) Methylation of DNA Target sites of methylation are - The cytidine

residues which exist as a dinucleotide, CG (written as CpG i.e Cytosine bound to guanine by phosphodiester bond).

↑methylated cytidine -- ↓Transcriptional activity

7

2.Regulation of Transcription• The differences in the mechanisms by which

the transcription of gene is controlled in prokaryotes and eukaryotes are listed below:

Prokaryotes Eukaryotes

The linked genes are organized into clusters known as operons which are under the control of a single promoter.

Eukaryotic genes are not organized into operons and each of these genes requires its own promoter.

These genes are primarily regulated by repressors.

Regulation by repressors is very occasional and the primary role of regulation is played by the transcriptional activators known as transcription factors.

8

2.Regulation of Transcription

Prokaryotes Eukaryotes A promoter sequence which

controls an operon lies upstream of the operon.

Accessory or the regulatory proteins control the recognition of the transcriptional initiation sites

by RNA polymerases

Those genes which code

for a protein have a basic

structure consisting of:Exons – Gene sequences which encode for a polypeptideIntrons – These sequences will get removed from the mRNA before it gets translated.A transcription initiation site

Promoter sequences. A single operon gets transcribed into a polycistronic mRNA which can be translated into multiple proteins

Monocistronic mRNAs which can produce a single polypeptide are produced

9

10

2.Regulation of Transcription

PromotersThe region necessary to initiate

transcription.

Consists of short nucleotide sequence that serve as the recognition point for binding of RNA polymerase.

Located immediately adjacent to the genes they regulate.

2.1: Promoters

PromotersProkaryotes - There are two promoter elements or

DNA sequences which are 35 and 10 base pairs in length and seated upstream to the transcriptional initiation sites.

The consensus sequence present at -35 position is TTGACA -10 position is TATAAT. This is also termed as Pribnow-

box.Eukaryotes – There are two types of promoters

which are:Basal promotersUpstream promoters

11

2.2: Promoters

Basal promoter or core promoter -These promoters reside within 40bp upstream of the start site. These promoters are seen in all protein coding genes. Examples are CCAAT-boxes and TATA-boxes

1. TATA box The consensus sequence for TATA box is

TATAT/AAT/A It resides 20 to 30 bases upstream of the

transcriptional start site This is similar in sequence to the prokaryotic

Pribnow-box Proteins like TFIIA, B, C interact with this TATA box

12

2.3: Promoters

2. CCAAT-box The consensus sequence for this is

GGT/CCAATCT It resides 50 to 130 bases upstream of the

transcriptional start site Protein named as C/EBP

(CCAAT-box/Enhancer Binding Protein) binds this box

Upstream promoters - These promoters may lie up to 200bp upstream of the transcriptional initiation site. The structure of this promoter and the associated binding factors keeps varying from gene to gene

13

Promoters

Promoters for RNA polymerase II include:

TATA box,CAAT box,

GC box,& Octamer box.

Site Structure Importance TATA box 25-30 bp

upstream(from the initial

point of transcription

8 bp sequences composed only of T=A pairs.

Mutations in this sequence greatly reduce transcription

(Loosing the ability to bind to transcription factors)

CAAT box 70-80 bp upstream

(from the initial point of transcription

CAAT or CCAAT sequence.

Mutations in this sequence greatly reduce transcription

GC box 110 bp upstream(from the initial

point of transcription

GGGCGG sequence, often present in multiple copies.

Documented by mutational analysis

Octamer box 120-130 bp upstream

(from the initial point of transcription

ATTTGCAT sequence.

Affects the efficiency of promoter in initiating transcription.

3. Enhancers

DNA sequences interact with regulatory proteins

increase the efficiency of initiation

of transcription

increase its rate.

3.1:Enhancers:

1. Large ) up to several hundred bp long).

2. Tissue- specific ( stimulate transcription only in certain tissues).

3.2: Enhancers

EnhancersEnhancers can be located upstream, downstream or

within the gene that is transcribedThe binding of these enhancers with enhancer

binding proteins (transcription factors) increases the rate of transcription of that gene to a greater extent.

Promoters are capable of initiating lower levels of transcription.

Enhancers are responsible for the cell or tissue specific transcription.

Each enhancer has its own transcription factor that it binds to.

17

3.3: Enhancers1. The proteins that bind to enhancers affect the

activity of proteins that bind to promoters.

2. Enhancers may allow RNA polymerase to bind to DNA and move along the chromosome till it reaches a promoter site.

3. May respond to molecules outside the cell ( e.g : steroid hormones).

4. May respond to molecules inside the cell ( e.g : during development thus the gene participates in cell differentiation).

3.4:How enhancers can control transcription although they are located

away from the transcription site.

Enhancers bind to transcription factors by atLeast 20 different proteins

Form a complex

change the configuration of the chromatin

folding, bending or looping of DNA.

3.5:Action of an enhancer – An enhancer binding protein has two binding

sitesBinds DNA Binds the transcription factors that are bound to the

promoter

20

21

DNA looping will bring the distal enhancers close to the promoter site to form activated transcription complexes, then the transcription is activated, increasing the overall rate of RNA synthesis.

3.6:Enhancers:

4.Transcription factors

“ Are the proteins that are essential for initiation of the transcription, but they are not part of RNA polymerase molecule that carry out the transcription process”.

Function:

Each RNA polymerase requires a number of transcription factors which help in:

1. Binding of the enzyme to DNA template.

2. Initiation and maintenance of transcription.

3. Control the rate of gene expression.

Transcription factors

Structure & Mechanism of action

These proteins contain 2 functional domains, that perform specific function.

1. DBD: DNA binding domain: binds to DNA sequences present in regulatory regions (e.g : TATA binding protein).

2. AD: Transcriptional activating domain: activate transcription via protein-protein interaction

Types of transcription factors:

1. Basal transcription factors:

The initiation of transcription by RNA polymerase II requires the assistance of several basal transcription factors.

Each of these proteins binds to a sequence within the promoter to facilitate the proper alignment of RNA polymerase on the template strand of DNA.

The basal TFs must interact with the promoters in the correct sequence to initiate transcription effectively.

TFIID is the 1st basal TF that interact with the promoter ; it contains TATA- Binding Protein.

Followed by TFII B, F, E, H & J.

Types of transcription factors:

2. Special TFs:Involved in regulation of heat, light, and hormone

inducible genes.They bind to:a. enhancers.b. Basal TFs.c. RNA polymerase that bind to the gene promoter.Therefore, special TFs can regulate the

transcriptional activity of the gene.

Types of transcription factors:

How is the gene transcription controlled at this point

• The unique combination of the promoter sites, transcription factors and enhancers chosen ultimately decides which gene gets switched on and which one gets switched off.

33

5.Regulation of RNA Processing

RNA processing involvesAddition of 5' capAddition of a 3' poly (A) tailRemoval of introns

The RNAs which get translated to proteins are transported out from the nucleus to cytoplasm.

Depending on the final combination of exons after splicing different kinds of proteins are obtained which can perform different functions in the cell.

34

5.1:Exon Shuffling

• The functions of two proteins synthesized from the same mRNA are different in different cells as different combination of exons exist in different cells.

35

6.Regulation of RNA Transport

• Only some RNAs function within the nucleus whereas all other RNAs which are meant for protein synthesis have to be transported from the nucleus to the cytoplasm via nuclear pores.

36

6.1:Regulation of RNA Longevity

• mRNAs from different genes have different life spans.

• The information of the life span of mRNA is found in the 3' UTR(Un-translated Reagion).

• The sequence AUUUA within 3' UTR acts as a signal for early degradation.

• More the number of times the sequence is repeated Shorter the lifespan of mRNA

37

38

three prime untranslated region (3' UTR)

6.1:Regulation of RNA Longevity

6.Regulation of Translation

Translational initiationThe expression of a gene product also depends

on the ability of the ribosome to recognize the correct AUG codon out of the multiple methionine codons present in the mRNA.

Control of translational process In many animals large amounts of mRNAs are

produced by the eggs but all of them do not get translated until the egg is fertilized.

39

7.Post Translational Control Points

Post translational modificationsFunctional state of protein depends on

modifications like glycosylation, acetylation, fatty acylation, disulfide bond formations.

Chaperons Protein transport

Transportation to the site of action also regulate gene expression.

Protein stabilityThe lifespan of a protein depends on the specific

amino acid sequence present within them

40

Summary of the Class

• The expression of genes is controlled at various levels in eukaryotes.

• At the chromatin stage the level of condensation determines whether the genes will remain transcriptionally active or not.

• The unique combination of the promoter sites, transcription factors and enhancers regulates the transcriptional rate of a gene.

• After transcription the gene expression is controlled by RNA processing.

• The expression of gene is also controlled at the level of translation and after translation.

41

42

THANK YOU