molbiol regulation

8/10/2019 MolBiol Regulation

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Control of Gene Expression


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Gene expression

Gene: The region of DNA that controls a discrete hereditary characteristic of

an organism, usually corresponding to a single protein or RNA.

Gene expression: Production of an observable molecular product (RNA or

protein) by a gene.


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Genes can be expressed with different efficiency

- Many identical RNA copies can made from the same gene, and each RNA

molecule can direct the synthesis of many identical protein molecules.

- Each gene can be transcribed and translated with a different efficiency. Gene A is

transcribed and translated much more efficiently than is gene B. This allows the

amount of protein A in the cell to be much higher than that of protein B.


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The different cell types of a multicellular organism

contain the same DNA

Both of these cells contain the samegenome, but they express different

RNAs and proteins


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A differentiated cell contains all the genetic instructions

necessary to direct the formation of a complete organism

- The nucleus of a skin cell from an adult frog transplanted into anenucleated egg can give rise to an entire tadpole.

- Cell differentiation generally depends on changes in gene expression rather

than on any changes in the nuleotide sequence of of the cellsgenome


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Evidence that a differentiated cell contains all the

genetic instructions necessary to direct the formation of

a complete organism

In many types of plants, differentiated cells retain the ability to

dedifferentiate, so that a single cell can form a clone of

progeny cells that later give rise to an entire plant


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Gene expression can be regulated at many of the steps in

the pathway from DNA to RNA to protein

The genome of a cell contains in its DNA sequence the information to make

many thousands of different protein and RNA molecules. A cell typically

expresses only a fraction of its genes, and the different types of cells inmulticellular organisms arise because different sets of genes are expressed.

Moreover, cells can change the pattern of genes they express in response to

changes in their environment, such as signals from other cells. Although all of

the steps involved in expressing a gene can in principle be regulated, for most

genes the ini tiation of RNA transcription is the most important point of control.


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Transcription is controlled by regulatory proteins

binding to regulatory DNA sequences

Regulatory proteinsare DNA-binding proteins that recognize

specific sites at or near the genes they control.

Activators increase transcriptionof the regulated genes (positive

regulators)

Repressorsdecrease or eliminatethe transcription of the regulated

genes ( negative regulators)


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Transcriptional regulation

in Prokaryotes


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The phases of the transcription cycle:

Initiation, Elogation, and Termination


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Features of bacterial promoters

- There are two conserved sequences: -35 and -10 regions (or elements), each of six

nucleotides, are separated by a nonspecific stretch of 17-19 nucleotides.- An addition DNA element that binds RNA polymerase is found in some strong promoter, for

example those directing expression of the rRNA genes. This is called UP-element and

increase polymerase binding by providing an additional specific interaction between the

enzyme and the DNA

- Some promoters lack a -35 region and instead has a so-called extended -10 element. This

comprises a standard -10 region with an additional short sequence element at its upstream end.

TTGACA TATAATStart site


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repressor

Activation by recruitment of RNA polymerase

(a) In the absence of both activator

and repressor, RNA

polymerase binds the promoter

and initiate a low level (basal

level) of transcription.(b) Binding of the repressor to the

operator sequence blocks

binding of RNA polymerase

and so inhibits transcription

(c) Recruitment of RNApolymerase by the activator

gives high levels of

transcription.


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Allosteric activation of RNA polymerase

(a) Binding of RNA

polymerase to promoter in

a stable close complex

(b) The activator interacts

with polymerase to trigger

transition to the open

complex and high levelsof transcription


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Action at a distance and DNA looping

DNA-bending protein: A protein that bends binding sites closertogether in space and thereby helps the interaction between the

DNA-bound activator and RNA polymerase


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Regulation of transcription initiation:

example from bacteria

- Operon: a set of genes that is transcribed in to a single mRNA (polycistronic mRNA)

- The lacoperon: The genes involved in lactose metabolism inE. coli. The lacoperon

consists of three lac genes: LacZ, lacYand lacA

-Thelacpromoter, located at 5end of lacZ, directs transcription of all three genes as

a single mRNA. This mRNA is translated to give three protein products. The LacZ

gene encodes the enzyme -galactosidase which cleaves the sugar lactose intogalactose and glucose. The lacY gene encodes the lactose permease, a protein that

inserts into the cell menbrane and transports lactose in the cell. The lacAgene encodes

thiogalactoside transacetylase which rids the cell of toxic thiogalactosides

- TheCAP site and the operator are ech about 20bp. The operator lies within the

region bound by RNA polymerase at the promoter, and the CAP site lies just upstream

of the promoter


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Expression of thelac genes

repressor

- Activator: CAP (Catabolite

Activator Protein)

- Repressor: Lac repressor

- Glucose and lactose levels

control the initiation of

transcription of the lacoperon through their effects

on the lac repressor and

CAP.


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- NtrC controls expression of genes involved in nitrogen metabolism, such as

theglnAgene- NtrC binding site located some 150bp upstream of the promoter

- There is a binding site for another protein, called IFF, located between the

NtrC binding site and the promoter. Upon binding, IHF bends DNA. By

bending the DNA, IHF bring the DNA-bound activator closer to the promoter,

helping the activator interact with the RNA polymerase bound there.

Activator

binding

site

Promoter

glnA

NtrC

IHF

DNA-bending

protein

NtrC: Transcriptional activator that work by allostery

rather than by recruitment


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Activation by NtrC

- RNA polymerase is prebound to the promoter in a stable closed complex.

- The activator NrtC has an enzymatic activity (ATPase) that induces a conformational

change in polymerase, triggering transition to the open complex. Thus the activating event

is an allosteric change in RNA polymerase.

- DNA-bending protein (IHF) can facilitate interaction between DNA-binding proteins.


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Transcriptional regulation

in Eukaryotes


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Transcription in prokaryotes and eukaryotes

Prokaryotes Eukaryotes

- Bacteria have only one RNA

polymerase

-Eukaryotes have three RNA polymerases:

RNA Pol I, RNA Pol II, and RNA Pol III (Pol

II is responsible for protein-coding genes; Pol

I transcribes the large ribosomal RNA

precursor gene; Pol III transcribes tRNAgenes, some small nuclear genes, and the 5S

rRNA gene.

- Bacteria require only one

additional initiation factor

(sigma factor) that mediates

binding of polymerase to thepromoter

- Several initiation factors are required for

efficient and promoter-specific initiation in

eukaryotes. These are called the general

transcription factors(GTFs)- Rather, additional factors are required,

including the so-called Mediator complex,

DNA-binding regulatory proteins, and

chromatin-modifying enzymes


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RNA polymerase II core promoter

- BRE: TFIIB recognition element

- TATA box

- Inr: Initiator element- DPE: downstream promoter element


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RNA polymerase II forms a pre-initiation complex with

general transcription factors at the promoter


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Overview of transcriptional control in Eukaryotes

- Nucleosomes and their modifiers influence access to genes. Eukaryotic cells contain

number of enzymes that modify histones; these modifications affect the transcriptional

machinery.

- Many eukaryotic genes have more regulatory binding sites and are controlled by more

regulatory proteins than are typical bacterial genes.


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The regulatory elements of a bacterial, yeast and

human gene

The increasing complexity of regulatory sequences from a simple

bacterial gene controlled by a repressor to a human gene

controlled by multiple activators and repressors.


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Eukaryotic genes are controllded by multiple

regulatory proteins


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Eukaryotic gene regulatory proteins control gene

expression from a distance

- The DNA sites to which the eukaryotic gene activators bound are original termed enhancer.

- Additional proteins serve to link the distantly bound gene regulator proteins to the RNA

polymerase and general transcription factors; the most important is a large complex of protein

known as the mediator.


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Mediator consists of many subunits, some conserved

from yeast to human


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Nucleosomes are the building blocks of chromosomes

-The nucleosome composed of a core of 8 histone

proteins (histone octamer)- two molecules each of

histone H2A, H2B, H3, H4- and the DNA

warapped around them.- The DNA between each nucleosome is called

linker DNA (20-60 bp).

- the DNA most tightly associated with the

nucleosome is called core DNA (146 bp)


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Structure of core histones

- A conserved region found in every core

histone , called the histone-fold domain.

- Histone fold is composed of three

helical regions separated by two short

unstructured loops (fig. B)- The core histonea each have an N-terminal

extension, called N-terminal tailbecause

it lacks a defined structure and is accessible

within the intact nucleosome.


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Nucleosome remodeling complexes

- Nucleosome remodeling complexes:

protein machines that use the energy of

ATP hydrolysis to change the structure

of nuclesomes- Different nucleosome remodeling

complexes disrupt and re-form

nucleosome, to allow increased access

to the DNA. The DNA-binding protein

could be involved in gene expression,

DNA replication, or DNA repair.


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Some prominent types of covalent amino acid side-chain

modifications found on nucleosomal histones


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Modification of the N-terminal tails of the histones

alters the function of chromatin

Me

Ac

P Phosphate group

Acetyl group

Methyl group


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Modification of the N-terminal tails of the histones

alters the function of chromatin


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Eukaryotic gene regulatory proteins can direct local

alterations in chromatin structure


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Transcription repressors

Transcription of eukaryotic genes can be

repressed in various ways:

- By binding to a site on DNA that overlaps

the binding site of an activator, a repressor

can inhibit the binding of activator to a

gene.

- A repressor binds to a site on DNA beside

an activator and interacts with thatactivator, occluding its activating region,

- A repressor binds to a site upstream of a

gene, by interacting with the

transcriptional machinery at the promoter

in some specific way, inhibit transcriptioninitiation.

- Repression by recruiting histone modifiers

that alter nucleosome in ways that inhibit

transcription


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Switching a gene off through DNA methylation and

histone modification


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Summary

- The transcription of individual genes is switched on and off in cell by

gene regulation proteins. These act by binding to short stretches of

DNA called regulatory DNA sequences

- In bacteria, regulatory protein usually binds to regulatory DNA

sequences close to where RNA polymerase binds and then either

activate or repress transcription of the gene. In eukaryotes, these

regulatory DNA sequences are often separated from the promoter by

many thousands of nucleotise pairs

- Eukaryotic gene regulatory protein act in two fundamental ways: (1)

they can directtly affect the assembly process or RNA polymerase

and general transcription factors at promoter, and (2) they can locallymodify the chromatin structure of promoter region

- In eukaryotes, the expression of a gene is generally controlled by a

combination of gene regulatory proteins.

molbiol regulation

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