GeneExpPF

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Gene Expression

and

Regulation

Course GTB 204/3

Dr. Mohammed Shahjahan

Reference Books

1. Principles of GeneticsD.P. Snustad and M.J. SimmsonJohn Willy & Sons, Inc, publishers, New York

2. Molecular Cell BiologyH. Lodish, A. Berk,S.L. Zipursky, P.Matsudaria, D. Baltimore

and J.Darnel. W.H. Freeman & Company publishers, New York

3. Principles of GeneticsRobert H. Tamarin, Mc Graw Hill publishers, Boston, USA

Syllabus outline

Know the Gene expression & Regulation

Overview of gene expression

Exons

Introns

mRNA-splicing

Transcription terminator

Overview

of DNA and

gene

structure

An eukaryotic gene

structure. In

prokaryotes

there is no intron.

(Ref. DNA Technology, I.E.

Alcamco,chap-3)

Central dogma

(Ref. Principle of Genetics,

Snustad & Simmons, chap-12)

Comparison of gene expression in

prokaryotes and eukaryotes

Prokaryotes Eukaryotes

Simple Complex

Genes grouped in Operons Genes have Introns and Exons

Polycistronic-Multiple genes Monocistronic-Single gene

mRNA 5-cap and 3-polyA

No Post-translational Post-translational modifications

modifications e.g. glycosylation

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Gene organization in prokaryotes

and eukaryotes

Insert picture

(Ref. Mol. Cell Biology

Lodish & Darnel, chap-9)

Prokaryotic polycistronic mRNA

(Ref. Princ. of Genetics, R.T.

Tamarin, chap-11)

A typical prokaryotic promoter



Promoters in prokaryotes and

eukaryotes



Eukaryotic promoter

Comparison of prokaryotic & Eukaryotic promoters

EukaryotesProkaryotes

1.At -10 position a conserved

sequence TATAAT called

Pribnow orTATA box is

present

2. At -35 position a conserved

sequence TTGTCA is present

3. Both -10 & -35 conserved

sequences are recognised by

sigma factor of RNA ploymerase

4. Another conserved sequence

centered at -50 called upstream

element is found in ribosomal

RNA

1. All the three eukaryotic RNA

ploymerases recognizes the conserved

sequence TATAAA (TATA boxat about -30 on the promoter DNA.

2 A second conserved sequence is

called CAAT box usually occurs at -80and the consensus seq is GCCAATCT

3 Two other conserved seq the GC box,

GGGCGG and the octamer box,

consensus ATTTGCAT are often

present in RNA pol II promoter

3. RNA pol II interact with several

proteins called transcriptional factors

(TF) in order to attack the promoter.

4. Activators (Enhancers) are also bind

to DNA seq often 100-1000bp

upstream of promoter.

Binding of RNA ploymerase II with eukaryotic

promoter

(Ref. Prin. of Genetics,

R.H. Tamarin, chap-10)

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RNA

synthesis Gene

Expression

overview



Initial steps in gene

expression

Gene expression and

regulation

in prokaryotes

What is an operon

We can define an operon as a sequence of adjacent genes all under

The transcriptional control of the same promoter and operator.

PromoterThe region on DNA with which RNA polymerase binds immediately

before beginning transcription is known as a promoter. Promoter

have the information for transcription initiation and the major sites in

which gene expression is controlled.

OperatorOperator or Operator site is a control element (receptor site)

sequence of DNA that is recognized by the end product of regulator

gene, repressor. Binding of repressor protein at operator exert its

influence over transcription.

Inducible and Repressible operon

Inducible operon

The inducible operons are activated when the substrate thatis to be catabolized enters the cell. E.g. lac operon.

Repressible operon (Anabolic operon)

Repressible operons are are turned off (repressed) when

their end product accumulates in excess of the needs of the

cell. E.g. trp operon

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lacoperon

P/O

P; promoter, O; operator

(Ref. Mol. Cell Biology, Lodish & Darnell, chap-10)

lacoperon with control region

(inducible system)

(Ref. Prin. Of Genetics by R. Tamarin Chap-10

lacoperator sequence


Jacob &

Monod model

of

transcriptional

regulation

(Ref. Mol. Cell

Biology, Lodish &

Darnell, chap-10)

lacoperator

(Oc)

mutantsare

cis-acting

(Ref. Mol. Cell Biology, Lodish &

Darnell, chap-10)

Cis-acting; regulatory

seq in DNA (promoter)

that can control a gene

only on the same

chromosome

lacl+ gene is trans-acting


Trans-acting: DNAseqencoding

diffusible proteins that control genes

on the same or differentchromosomes

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lacoperon inducer IPTG


Induction of

lacoperon

E.coli growing on glucose

Induction with IPTG

Addition of3H-uridine

Cell lysis & RNA isolation

Hybridization with lac

DNA


Catabolite repression oflacoperon

An interesting property of the lac operon and other operons that

code for enzymes that catabolize sugars ( e.g. arabinose and

galactose operons) is that they are all repressed by the presence

of glucose. That is glucose is cataboilized in preference to other

sugars this is called catabolic repression.

Catabolic repression involves cyclic AMP. cAMP work in

conjunction with another regulatory protein, the catabolic

repressor protein (CAP) to control the transcription of certain

operons

ATP Adenylcyclase Cyclic AMP CAP protein

( inhibited by glucose) control of

transcription

Positive & negative transcriptional

control oflacoperon

Catabolite repression

(Ref. Mol. Cell Biology, Lodish &Darnell, chap-10)

Trp operon

(repressible

system)

(Ref. Prin. Of Genetics by R.

Tamarin Chap-13)

Repression in trp operon

(Ref. Prin. Of Genetics by R. Tamarin Chap-13)

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Conc epts

* Proka ryote s exhib it efficient gen etic m echan isms to respond to

env ironmen tal condi tion s

* Lacto se metabol ism i n E. coli is regulated by an inducib le

system* The Catabol ite A ctiv ating Prote in (CAP) exerts positive

con trol ov er

the lac ope ron

* Crystal structure analy sis of repressor comp lexe s has

con firmed the

ope ron mode l

* The tryptophan ope ron in E. coli is a repressibl e gen e system

* Attenuat ion is a critical process during the r egula tion o f th e trp

ope ron

* The ara ope ron is cont rolled by a regul ator protein tha t exerts

both

positive and negative c ont rol

Gene expression in Bacteria

Gene

Expression andRegulation

in Eukaryotes

Gene tic regul ation in euk aryote s can occur at seve ral levels ,

but transcription al cont rol is the p rimary m echan ism con troll ing

gen e exp ression . Tr anscription is modu lated by the int eraction of

regu lato ry

mo le cu le s wi th sh o r t DN A se qu en c e s mo s t o f te n lo c a te d u p s t r e a m

from affected

g en e s . P o s t t r a nsc r ipt ion al me c h an i sms inv olve th e s e l e c t ion o f

al ternativ e

produc ts from a sing le t ranscript and the con trol of mR NA

stabili ty.

Con c e p t s

* Euk a ryo ti c g en e r e gu lat io n i s v e ry d i f f e r en t f ro m p ro c a ryot i c

g en e

regu lation

* The promo ter is th e si te of assembly o f the ba sal transcription

com plex

* Enh a n ce r s c o nt rol c h rom a t in s t ruc tu re a n d th e r a t e o f

transcript ion

* Th e ye a s t g a l g e n e s a r e p o s i t i ve - in du c ibl e a n d c a ta bo l it e -

repressible

* D NA m ethyl ation can a ffect gene regul ation

* P ost- tran scription al regula tion includ es a lternate sp licing andme ssa g e

stab il i ty

GENE EXPRESSION IN EUKARYOTESStages of

eukaryotic

gene

expression

(Ref. Prin. Of Genetics by

Snustad & Simmons,

chap-24)

m-RNA Splicing

In eukaryotes the exons ( protein coding regions) are interrupted by

introns (non-coding region).

The primary transcripts or pre-mRNAs in eukaryotes often must be

processed by the excision of introns and the addition of 5-methylguanosine caps (MG) and 3-poly (A) tails. The post-transcriptional

regulation includes alternate splicing and message stability.

The processed mRNA is then transported to cytoplasm for protein

syntheis (translation).

Post-

transcriptional

processing in

eukaryotes



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RNA

splicing



Self-splicing of

Tetrahymena thermophilia

rRNA precursor



Alternate splicing

(Ref. Prin. Of Genetics by Snustad & Simmons, chap-24)

Heat shock induction ofDrosophila

hsp70gene



Regulation of gene expression by

steroid hormone



Regulation of gene expression by

peptide hormone



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Structural

motifs within

transcriptional

factors



Upstream activating sequences in

Gal1 and Gal10genes in yeast



Transcription terminator

1. Rho-independent terminators

Rho-independent termination cause termination of transcription

even if rho is not present.

2. Rho-dependent terminators

Rho-dependent terminators require the rho-protein;without it RNA

polymerase continues to transcribe, pass the terminator, called

read through.

There are 2 -types of terminators, rho-dependent and rho-independent.

Difference lies in their dependency on a protein called rho protein. Rho

() is a hexamer protein (six identical copies).

(Ref. Princ. of Genetics, R.T. Tamarin, chap-10)

Terminator regions in DNA

(Ref. Princ. of

Genetics, R.T.

Tamarin, chap-10)

Transcriptional terminator (rho-

independent)



rho-dependent & rho-independent

termination

(Ref. Principle of

Genetics, R.T.

Tamarin, chap-10)

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