IMPORTANCE AND FUNCTIONS OF GENE EXPRESSION

Regulation is an important aspect of almost every process in nature. Rarely, if ever, it is

adequate simply to describe the steps of a process. Instead, we must also ask what

turns it on, what turns it off, and what determines its rate. Most genes are not expressed

all the time. In some cases, selective gene expression enable cells to be metabolically

thrifty, synthesizing only those gene products that are of immediate use under the

prevailing environmental condition, this is often the situation with bacteria. In other

cases, such as in multicellular organism, selective gene expression allows cells to fulfill

specialized roles.

Bacterial Gene Regulation

Of the several thousand genes present in a typical bacterial cell, some are so important

that they are always active in growing cells. Examples of such constitutive genes

include ribosomal genes and genes encoding the enzymes of glycolysis. For most other

genes, expression is regulated so that the amount of the final gene product (protein or

RNA) is carefully tuned to the cell’s need for that product. A number of these regulated

genes encode enzymes for metabolic processes that unlike glycolysis, are constantly

required. One way of regulating the intracellular concentration of such enzymes is by

starting and stopping gene transcription in response to cellular needs. Because this

control of enzyme-coding genes helps bacterial cells adapt to their environment, it is

commonly referred to as adaptive enzyme synthesis.

CATABOLIC AND ANABOLIC PATHWAYS ARE REGULATED THROUGH INDUCTION AND REPRESSION

Bacteria use somewhat different approaches for regulating enzyme synthesis,

depending on whether a given enzyme is involved in a catabolic (degradative) or

anabolic (synthetic) pathway. The enzymes that catalyze such pathways are often

regulated coordinately, that is the synthesis of all enzymes involved in a particular

pathway is turned on and off together :

Catabolic pathways and substrate induction. Catabolic enzymes exist for the

primary purpose of degrading on specific substrates, often as a means of obtaining

energy such as the breakdown of the disaccharide lactose involve enzyme (β-

galactosidase) to galactose and glucose.

Anabolic pathways and end-product repression. The regulation of anabolic

pathways is in a sense just the opposite of that for catabolic pathways. The amount of

enzyme produced by a cell produced by a cell usually correlates inversely with the

intracellular concentration of the end-product of the pathways. For example, as the

concentration of tryptophan rises, it is advantageous for the cell to economize on its

metabolic resources by reducing its production of the enzymes involved in the

synthesizing tryptophan. But it is equally important that the cell be able to turn the

production of these enzymes back on when the level of tryptophan decrease again. This

kind of control is made possible by the ability of the end-product of an anabolic pathway.

For example, tryptophan, to somehow repress (reduce or stop) the further production of

the enzyme involved in this formation. Such reduction in the expression of the enzyme-

coding genes is called end-product repression. Repression is a general term in

molecular genetics, referring to the reduction in expression of any regulated gene.

Effector molecules. One future common to both induction and repression of enzyme

synthesis is that control is exerted at the gene level in both cases. Another shared

future is that control is triggered by small organic molecules present within the cell or in

the cell’s surroundings. Geneticists call small organic molecules that function this way

effectors. Effectors induce shape changes allosteric proteins that control gene

expression.

THE GENES INVOLVED IN LACTOSE CATABOLISM ARE ORGANISED INTO AN INDUCIBLE OPERON

The classic example of an inducible enzyme system occurs in the bacterium E. coli and

involves a group of enzymes involved in lactose catabolism. The genes involved in

lactose metabolism are lacZ gene, lacY gene and lacA gene that are lie next to each

other in the bacterial chromosome and are repressed only when an inducer such as

lactose present. lacZ gene codes for β-galactosidase that hydrolyzed lactose , lacY

gene codes for galactosidase permease (the plasma membrane protein that transport

lactose into the cell) and lacA gene codes for a transacetylase that adds an acetyl group

to lactose as it is taken up by the cell.

Operon is a group of genes with related functions that are clustered together with DNA

sequences that allow the genes to be turned on and off simultaneously.

The lac Repressor IS AN ALLOSTERIC PROTEIN WHOSE BINDING TO DNA IS CONTROLLED BY LACTOSE

For induction to occur, an additional gene must be present that is a regulatory gene

named lacI. lacI gene codes for a product that normally inhibits, and thereby regulates,

expression of the lacZ gene, lacY gene and lacA genes. A regulatory gene product that

inhibits the expression of other gene is called a repressor protein.

The lac operon that consist of lacZ , lacY and lacA genes preceded by a promoter and

a special nucleotide sequence called the operator, which actually overlaps the

promoter.Transcription begins at the promoter, which the site of RNA polymerase

attachment. The repressor protein, called the lac repressor, is encoded by lacI

regulatory gene, which located outside the operon. When the repressor is bound to the

operator, RNA polymerase is blocked from moving down the lac operon. In other words,

binding of the repressor to the operator keeps the operon’s genes turned off.

If binding of the repressor to the operator blocks transcription, how do cells turn on

transcription of the lac operon, as occurs in the presence of the inducers such lactose?

The answer is that the inducer molecules binds to the lac repressor, thereby altering its

conformation so that the repressor loses the ability to bind the operator site in DNA.

Once the operator site is no longer blocked by the repressor, RNA polymerase can bind

to the promoter and proceed down the operon, transcribing the lacZ , lacY and lacA

genes into a single polycistronic mRNA molecule.

THE REVELATION OF LAC OPERON ORGANIZATION

The studies of mutant bacteria bring to the revelation on how the lac Operon organised.

It involved the genetic analyses of mutant bacteria either produced abnormal amount of

enzyme of lac Operon OR showed abnormal responses to the addition or removal of

lactose. The mutation found to be located at Operon genes (lacZ, lacY or lacA) or

regulatory elements of system (O, P lac or lacI). These mutation can be distinguished as

mutation at operon genes only affect single protein, whereas, the mutation in regulatory

regions affect all expressions.

Based on the data obtained, the wild-type cells make small amounts of the lac enzyme

because the binding of repressor to operator is reversible. The active repressor

occasionally “falls off” the operator. The resulting low,, background level of transcription

is important because it allows cell to produe enough galactoside permease. Galactoside

permease facilitate the initial transport of lactose molecules into the cell priorto induction

of the lac operon.

Line Number

Genotype of Bacterium*

Phenotype with lnducer Absent

Phenotype with Inducer Present

B-galactosidase

Permease B-galactosidase

Permease

1 I+ P+ O+ Z+ Y+

- - + +

2 I+ P+ O+ Z+ Y-

- - + -

3 I+ P+ O+ Z.- Y+

- - - +

4 I+ P+ O C Z+ Y+

+ + + +

5 I+ P- O+ Z+ Y+

- - - -

6 IS P+ O+ Z+ Y+

- - - -

7 I- P+ O + Z+ Y+

+ + + +

As the experiment conducted, there are several operon model suggested. There are:

a) Operon Gene MutationMutations in lacy and lacZ can lead to the enxyme alteration with little or no

biological activity, even with the presence of inducer. The consequences of the

mutations is the unavailability to utilize lactose because there will be no

glycosidic bond between glucose and galactose or the regulatory sequence is

defective.

b) Operator MutationIt leads to the a constitutive phenotype. Constitutive phenotype caused the

mutant cells will continuously secrete lac enzyme with the presence of inducer or

not. The effect is the DNA sequences are shifted making the the repressor failed

to recognize.

c) Promoter MutationIt decreased the affinity of the RNA polymerase for the promoter, caused the rate

of mRNA production decreased. However, the mRNA still can elongate when

RNA polymerase able to attach to the DNA.

d) Regulatory Gene MutationsIt caused the superrepressor mutants as the mutants fail to produce any one of

the lacI with the presence of any inducer present. Either the repressor molecule

in such mutants has lost its ability to recognise and bind the inducer but still can

recognise the operator, OR, it has high affinity for the operator regardless of

whether inducer is bound on it. Thus, the enzymes synthesized in all conditions.

The Cis-Trans Test Using Partially Diploid Bacteria

Cis-Trans test is used to distinguished two different kinds of constitutive mutants, OC

and I -. Jacob and Monod constructed partially diploid bacteria by inserting a second

copy of the lac portion of the bacterial genome into the F-factor plasmid of F+ cells. This

second copy could be transfer by conjugation into a host bacterium of any desired lac

genotype to create prtial diploids such as in the table below.

The mutation is said cis if the only genes affected are those physically linked to the

mutant locus. In this case, it is referring to a mutation whose influence is restricted to

genes located in the same physical copy of the lac operon. In contrast it is said to be

trans when the genes have the ability to affect the both copies of the operon. The Jacob

and Monod determine which copy is being expressed in any given cell population by

using partial diploid bacteria (contained defective Z gene and Y gene).

From this the table, if one of these lac operon copies have defective I+ and I- we know

that this occurs because the one functional I gene present in the cell produces active

reprresor molecules that diffused through the cytosol and bind to bath operator sites in

the absence of lactose. The repressor is now known as trans-acting factor.

If the partial diploid bacteia containing both the O+ and O- allele, the genes linked to the

Oc allele are constitutively transcribed (Z+Y-), and those linked to the wild type allele are

incrucible (Z-Y+). The O locus who are in cis action, affects the behaviour of the genes

only in the operon it is physically a part of. The O site is now known as cis-acting element.

Line Numb

er

Genotype of Diploid Bacterium"

Phenotype with lnducer Absent

Phenotype with Inducer Present

B- galactosida

se

Permease B- galactosida

se

Permease

1 I+P+O+Z+Y+/I+P+O+Z+ - - + +2 I+P+O+Z-Y+/

L+P+O+Z+Y-- - + +

3 I+P+O+Z-Y+/L-P+O+Z+Y- - - + +4 I+P +O +Z - Y+/L+ P+ OC

Z+ Y-+ - + +

5 I+P+O+Z- Y+/IS P+ O+ Z+ Y-

- - - -

The Genes Involved In Tryptohan Synthesis Are Organized Into A Repressible Operon

The tryptophan is a good example of repressible operon. The trp operon contains gene

coding for the enzymes involved in tryptophan biosynthesis, along with DNA sequences

that regulate the production of these enzymes. The product of this biosynthesis

pathway, the amino acid tryptophan.

The regulatory gene for this operon is, called trpR, codes for a trp repressor protein

that-incontrast to the lac repressor- is active (binds to the operator DNA) when the

effector is attached to it and is inactive in its free form. The effector in this system also

called as corepressor because it required, along with the repressor protein to shut off

transcription of the operon.

Figure 1.1 Regulation of The Tryptophan Operon by the trp repressor.

Catabolite Repression Illustrates the Positive Control of Transcription

Catabolite Repression is refer to the ability of the of glucose to inhibit the synthesis of

catabolic enzymes produced by inducible bacterial operons. Glucose does not effect the

expression of the genes, whereas, the second signalling that reflects the level of the

glucose, called AMP called cyclic AMP will control the gene expression. Glucose is

preferable because glucose is the sources of energy for almost cells as the enzymes

glycotic and TCA produced constitutively. Glucose acts by indirectly inhibiting adenylyl

cyclase, the enzyme that catalyzes the synthesis of cAMP from ATP. Thus, the more

glucose [resent, the less cAMP is made.

Camp Receptor Protein (CRP) is an activator proteins that will turn on transcription. By

itself, the CRP is non-functional. It needs to be complexed with cAMP. CRP changes to

an active shapes that enables it to bind to a particular base sequence within operons

that produced catabolic enzymes.

Figure 2 The cAMP Receptor Protein (CRP) and Its Functions.

When CRP in its active form (that is, the CRP-cAMP complex), attaches to one of its

recognition sites in the DNA, the binding of RNA polymerase to the promoter is greatly

enhanced, thereby stimulating the initiation of transcription.

When the glucose concentration inside the cell is high, the cAMP concentration falls and

hence CRP is largely in its inactive form. Therefore, CRP cannot stimulate the

transcription of the operons that produced catabolite enzymes. However, when the

concentration of the glucose falls, the cAMP rises, activating CRP by binding to it. The

catabolitic enzymes produced allow cell to obtain energy from the breakdown of the

nutrient other than glucose.

Inducible Operons Are Often Under Dual Control

Previously, we know that lac operon might be controlled in two ways, positive or

negative.

Negative control is based on repressor-operator interactions allow the presence of an

alternative energy sources to turn on the particular operon. Positive control system is

based on the action of CRP, makes transcription of the operon sensitive to the glucose

concentration in the cell, as mediated by the cAMP level.

If in binding to the DNA, the regulatory prevents or turns off transcription, then it is a part

of the negative control mechanism. Meanwhile, if its binding DNA results in the activtion

of transcription, then the regulatory protein is part of positive control mechanisms. In

response to the allosteric effector, the transcription of an inducible operon is activated

whereas the transcription of a repressible operon is inhibited.

Sigma Factors Determine Which Sets of Genes Can Be Expressed

Sigma is a kind of protein that recognized the gene promoter sequences. In E. coli, the

prevalent sigma factor is Ơ70. However, the environmental factors such as increase in

temperature, UV radiation or aciditity can trigger the use af alternative sigma factor.

When sigma factors bound to the RNA polymerase core enzyme, promoter recognition

is altered, thereby initiating the transcription of genes encoding proteins that help the

cell adapt to the altered environment.

Sigma factors, Ơ54 enables RNA polymerase to prefentially transcribed genes whose

products minimize the slowing of growth under nitrogen-limiting factor. Each bacteria

has different number of sigma factors. It is presumably control subsets of genes in

response to different environmental signals and cellular conditions.

Figure 3 The diagramatic sigma factor.

Figure 4 The mechanisms of sigma factor.

Attenuation Allows Transcription to Be Regulated After the Initiation Step

Figure 5 The trp mRNA Leader Sequence

This leaders has two unusual features that enable it to play a regulatory role. First in

contrast to the untranslated leader sequences typically encountered at the end 5’ end of

mRNA molecules, a portion of the trp leader sequence is translated, forming a leader

peptide 14 amino acid long. Within the mRNA sequence coding for this peptide are two

adjacent codons for the amino acid.

Second, the the trp sequence contains four segments (labelled 1,2,3 and 4) whose

nucleotides can base-pair with each other to form several distinctive hairpin loop

structure. The region 3 and 4 plus an adjacent string of eight U nucleotides is called

terminator. The hairpin loops are acting as a transcription terminal single.

The translation of leader RNA plays a crucial role in attenuation. A ribosomes attached

to its first binding site on the trp mRNA , it follows behind the RNA polymerase. When

level of tryptophan is low, the level of tryptophanyl-tRNA also low. Thus, when the

ribosomes arrive at the tryptophan codons of the leader RNA, it stalls briefly.

The stalled ribosomes blocks region 1, allowing the alternative hairpin structure to form

by pairing region 2 and 3. When region 3 in this tied, it cannot form terminator with

region 4 causing the transcription by RNA polymerase continues, eventually producing

a complete mRNA transcript of trp operon. The ribosomes used the mRNA to

synthesize the tryptophan enzymes pathway, causing its level increased.

If the tryptophan is plentiful and the tryptophanyl-tRNA levels are high, the ribosome

does not stall at the tryptophan codons and eventually region 2 is blocked. This pause

permits the formation of the 3-4 hairpin, which is the transcription terminal signal.

Transcription by RNA polymerase is therefore terminated near the end of the leader

sequence, and mRNAs coding for the enzymes are no longer produced.

The probability of terminating versus continuing transcription is highly sensitive to the

small changes in the concentration of tryptophanyl-tRNA , providing a more responsive

control system than can be achieved by the interaction of free tryptophan with the trp

repressor as the only means of regulations.

Riboswitches Allow Transcription and Translation to Be Controlled by Small Molecule Interaction with RNA

The ability of small molecules to induce shape changes in allosteric proteins plays a

central role in regulating gene expression. For example, how gene transcription is

controlled by the binding of allolactose to the lac repressor protein, tryptophan to the trp

repressor protein and cAMP to the cAMP receptor protein. In each case, mRNA

production is altered by the binding of small molecule to a regulatory protein.

Small molecule can also regulate gene expression by binding to special site in mRNA

called riboswitches. Binding of an appropriate small molecule to its corresponding

riboswitch triggers changes in mRNA shape that can affect either transcription or

translation. Riboswitches are typically found in the untranslated leader region of mRNA

transcribed from bacterial operons, although they have been detected in mRNAs from

archaea and eukaryotes as well. One of the first riboswitches to be discovered is in the

RNA transcribed from the riboflavin (rib) operon of the bacterium B.subtilis.

The rib operon contains five genes coding for enzymes involved in synthesizing the

essential coenzymes, FMN and FAD. RNA transcribed from the rib operon possesses a

leader sequence that like the trp leader which can fold into hairpin loop that terminates

transcription. As shown in the figure 2, binding of FMN to the leader sequence

promotes formation of this hairpin loop. Transcription of the rib operon is therefore

terminated when FMN is present, halting the production of enzymes that are not

necessary because they are involved in synsthesizing FMN itself.

Figure 2:

Besides regulating transcription, the binding of small molecule to riboswitches can also

control mRNA translation. For example occurs in mRNAs coding for enzymes involved

in the pathway for synthesizing FMN and FAD in a single operon, but the some of them

are still controlled by riboswitches. In this case, binding of FMN to its mRNA riboswitch

promotes the formation of a hairpin loop that includes the sequence required for binding

the mRNA to ribosome. By sequestering away these sequence, the bound FMN

prevents the mRNA from interacting with ribosomes and initiating translation. (as shown

in figure 2b above).

Eukaryotic Gene Regulation: Genomic Control.

The DNA of eukaryotic cells is package into chromatin fibres and located in a nucleus

that is separated from the protein synthetic machinery by a nuclear envelope.

Eukaryotic genome are usually much larger than those of bacteria, and multicellular

eukaryotes must create a variety of different cell types from the same genome. Such

differences require a diversity of genetic control mechanisms that in some cases differ

significantly from those routinely observed in bacteria. Such different also indicate that

selectively controlling the expression of a wide variety of different genes must play a

central role in the mechanism responsible for creating differentiated cells.

Eukaryotic Gene Expression Is Regulated at Five Main Levels

The fact that a multicellular plant or animal may need to produce hundreds of different

cell types using a single genome underscores the difficulty of explaining eukaryotic

gene regulation entirely in terms of known bacterial mechanisms

The pattern of genes being expressed in any given eukaryotic cell is ultimately reflected

in the spectrum of functional gene products which usually protein molecule but in some

cases is RNA. The overall pattern is the culmination of controls exerted at several

different levels. These potential control points are highlighted in figure below which

traces the flow of genetic information from genomic DNA in the nucleus to functional

proteins in the cytoplasm.

The control is exerted at five main levels. First is the genome, then transcription, RNA

processing and export from nucleus to cytoplasm, translation and lastly posttranslational

events. Regulatory mechanisms in the last three categories are all examples of

posttranscriptional control, a term that encompasses a wide variety of events.

DNA Rearrangements Can Alter the Genome

A few cases are known in which gene regulation is based on the movement of DNA

segments from one location to another within the genome, a process known as DNA

rearrangement.

Yeast Mating-Type RearrangementsIn the yeast, Saccharomyces cerevisiae, mating occurs when haploid cells of two

different mating types, called α and a, fuse together to form a diploid cell. Cells

frequently switch mating type, presumably to maximize opportunities for mating. They

do so by moving the alternative allele into the MAT locus. This process of DNA

rearrangement is called the cassette mechanism because the mating-type locus is like a

tape deck into which either the α or the a “cassette” (allele) can be inserted and “played”

(transcribed).

Figure below describes the yeast cassette mechanism in more detail. The MAT locus,

containing either the α or a allele, is located on yeast chromosome 3, approximately

midway between extra copies of the two alleles. The locus that stores the extra copy of

the α allele is called HMLα, and the locus with the extra copy of the a allele is called

HMRa. In switching mating type, a yeast cell makes a DNA copy of the other mating-

type allele, either HMLα or HMRa, and inserts this new copy into the MAT locus. Before

the new copy can be inserted, however, the existing DNA at MAT must be excised (by a

site-specific endonuclease) and discarded. Unlike the DNA sequence residing at the

MAT locus, the DNA sequences at HMLα and HMRa never change.

Antibody Gene Rearrangements

A somewhat different type of DNA rearrangement is used by lymphocytes of the

vertebrate immune system for producing antibody molecules. Vertebrates make millions

of different kinds of antibodies, each produced by a different lymphocyte and each

capable of specifically recognizing and binding to a different foreign molecule. The

problem is if every antibody molecule were to be encoded by a different gene; virtually

all of a person’s DNA would be occupied by the millions of required antibody genes. The

rearrangement process involves four kinds of DNA sequences, called V, J, D, and C

segments. The C segment codes for a heavy or light chain constant region whose

amino acid sequence is the same among different antibodies. The V, J, and D

segments together code for variable regions that differ among antibodies and give each

one the ability to recognize and bind to a specific type of region molecule.

Figure below show the DNA regions containing the various V, D, and J segments are

rearranged during lymphocyte development to randomly bring together one V, one D,

and one J segment in each lymphocyte. The DNA rearrangement process that creates

antibody genes also activates transcription of these genes via a mechanism involving

special DNA sequence called enhancers. Enhancers are located near DNA sequences

coding for C segments, but a promoter sequence is not present in this area so

transcription does not normally occur.

DNase I Sensitivity Provides Evidence for the Role of Chromatin Decondensation in Genomic Control

The absence of polytene chromosomes in most eukaryotic cells makes it difficult to

visualize chromatin decondensation in regions of active genes. One particularly useful

research tool is DNase I, an endonuclease isolated from the pancreas. In test tube

experiments, low concentrations of DNase I preferentially degrade transcriptionally

active DNA in chromatin. The increased sensitivity of these DNA regions to degradation

by DNase I provide evidence that the DNA is uncoiled.

Coactivators mediate the interaction between regulatory transcription factors and the RNA polymerase complex

Two basic principles govern the interaction between enhancers and the genes they

regulate.First ,looping of the DNA molecule can bring an enhancer into close proximity

with a promoter,eventhough the two lie far aparts in terms of linear distance along the

DNA double helix. Second ,a diverse group of coactivator proteins mediate the

interaction between activators bound to the enhancer and the RNA polymerase complex

associated with the promoter.

Several type of coactivators play roles in these interactions.Example,the enzyme

histone acetyltransferase(HAT) is a coactivator that adds acetyl group to histone

molecules and thereby promotes chromatin decondensation.Another group of

coactivators are involved in chromatin remodeling,that altering chromatin structure by

moving nucleosomes out of the way to give transcription factors access to their DNA

target sites in the promoter region of gene.lastly,a large multiprotein complex called

Mediator functions as a coactivators by serving as a “bridge” that binds to activators

proteins associated with the enhancer .

How such interactions can trigger gene activation.?

1.A group of activator proteins bind to their respective DNA control elements within the

enhancer ,forming a multiprotein complex called an enhanceosome.One or more of

these activators proteins cause the DNA to bend,creating a DNA loop that brings the

enhancer close to the core promoter.

2.The activators interact with coactivators such as chromatin remodeling proteins

(SWI/SNF) and histone acetyltranferase(HAT),which alter chromatin structure to make

the DNA in the promoter region more accessible.

3.The activators bind to Mediator,which facilitates the correct positioning of RNA

polymerase and the general transcription factors at the promoter site and thus allows

transcription to begin.

Multiple DNA control elements and transcription factors act in combination

Multiple DNA control elements and their regulatory transcription factors are involved in

controlling eukaryotic gene transcription has led to a combinational model for gene regulation.This model propose that a relatively small number of different DNA control

elements and transcription factors,acting in different combination,can establish highly

specific and precisely controlled patterns of gene expression in different cell types.In

addition,transcription of gene that encode tissue-specific proteins requires the presence

of transcription factors or combinations of transcription factors that are unique to

individual cell types.This model show how liver cells produce large amounts of proteins

such as albumin but would prevent significant production of these proteins in other

tissues,such as brain.

A combinatorial model for gene expression

The gene for the protein albumin,like other genes,is associated with an array of

regulatory DNS elements.Cell of all tissues contain RNA polymerase and the general

transcription factors,but the set of regulatory transcription factors avaible varies with the

cell type. As example, liver cells contain asset of regulatory transcription factors that

includes the factors for recognizing all the albumin gene control elements.When these

factors bind to the DNA ,they facilitate transcription of the albumin gene at a high level.

In brain cells,however,have a different set of regulatory transcription factors that does

not includes all those albumin gene.Consequently,in brain cells,the transcription

complex can assemble at the promoter,but not very efficiently.

Several common structural motifs allow regulatory transcription factors to bind to DNA and active transcription.

Regulatory transcription factors posses two distinct activities,the ability to bind to a

specific DNA sequence and the ability to regulate transcription.The domain that

recognizes and binds to a specific DNA sequence is called the transcription factors

DNA-binding domain,where as the protein region required for regulating transcription is

known as the transcription regulation domain. Unique types of the structure have also

been detected in the DNA –binding domains of transcription factors.In fact,most

regulatory transcription factors can be placed into one of a small number of categories

based on the secondary structure pattern or motif,that make up the DNA- binding

domain. There are several type of these DNA- binding motifs.

Helix-Turn-Helix Motif.

One of the most common DNA-binding motif,detected in both eukaryotic and prokaryotic

regulatory transcription factors.This motif consists of two α helices separated by abend

in the polypeptide chain.Although the amino acid sequence of the motif differs among

various DNA –binding proteins,the overall pattern is always the same,one α helix,called

the recognition helic,contains amino acid side chains that recognize and bind to specific

DNA sequences by forming hydrogen bonds with bases located in the major groove of

the double helix,while the second α helix stabilizes the overall configuration through

hydrophobic interactions with the recognition helix.

Leucine Zipper Motif.

The leucine zipper motif is formed by an interaction between two polypeptide

chains,each containing an α helix with regularly spaced leucine residues.Because

leucines are hydrophobic amino acids that attract one another,the stretch of leucines

exposed on the outer surface of one α helix can interlock with a comparable stretch of

leucines on the other α helix,causing the two helices to wrap around each other into coil

that “zippers” the two helices together.

Helix-Loop-Helix Motif.

The helix-loop-helih motif is composed of short αhelix connected by a loop to

another,longer α helix.Like leucine zippers,helix-loop-helix motifs contain hydrophobic

regions that usually connect two polypeptides,which may be either similar or different.

Zinc Finger Motif

Initially identified in a transcription factor for the 5s rRNA genes (TFIIIA),the zinc finger

DNA-binding motif consists of an α helix and atwo segment β sheet, held in place by the

interaction of precisely positioned cysteine or histidine residues with a zinc ion.The

number of zinc finger present per protein molecules varies among the transcription

factors that posses them,ranging from two fingers to several dozen.

DNA Response Element Coordinate The Expression Of Nonadjacent Genes

To coordinate the expression of such physically separated genes,eukaryotes employ

DNA control sequences called response element to turn transcription on or off in

response to particular environmental or developmental signal.Response element can

function either as proximal control elements or as components of enhancers.In either

case,placing the same type of response element next to gene resising at different

chromosomal locations allows these gene to be controlled together eventhough they are

not loactaed next to one another.

Steroid Hormone Receptors Act as Transcription Factors That Bind to Hormone Response Element

The phenomenon of coordinated gene regulation is illustrated by the behavior of steroid

receptor protein,which mediate the action of steroid hormones such as

progesterone,estrogen,testosterone and glucocorticoids.

These gene-spicific effects stem from the ability of steroid receptors to act as

transcription factors that bind to DNA sequences called hormone response elements.To

illustrate how this arrangement works with specific hormone, consider cortisolas an

example.Cortisol is a member of related group of steroid hormones called

glucocorticoids because they stimulate glucose production and are made in the adrenal

cortex.Normally the glucocorticoid receptor (GR) is located mainly in the cytosol,where it

is bound to members of the Hsp family of chaperone proteins.As long as it remains

associated with Hsp protein,GR is prevented from entering the nucleus and binding to

DNA.But the binding of cortisol to GR triggers the release of the Hsp proteins.The GR

molecule is then free to kove into the nucleus,where it binds to glucocorticoid response

elements wherever they may reside in the DNA.The binding of a GR molecule to

response element in turn facilitates the binding of secong GR molecule to the same

response element.

The resulting GR dimer activates transcription of the adjacent genes by recruiting

coactivators that promote assembly of the transcriptional machinery.This binding of two

GR molecule to the same DNA site occurs because the DNA sequence of the

glucocorticoid response contain an invereted repeat,that is two copies of the same DNA

sequences oriented in opposite direction.Although steroid hormone receptors usually

stimulated gene transcription,in a few cases they inhibit it.For example,the

glucocorticoid receptors binds to two types of DNA elements,one type associated with

gene whose transcription activated,the other with gene whose transcription is inhibited.

Transcriptional Control

Control of Transcription Factors by Protein Phosphorylation. Phosphorylation is the addition of phosphate groups. CREBs and STATs are examples of transcription factors activated by phosphorylation. The phosphorylation of these transcription proteins (CREBs & STATs) is catalyzed by cAMP (cyclic AMP).

CREB protein

CREB protein normally binds to DNA sequences called cAMP response element (CRE), which is located adjacent to the genes. Upon being phosphorylated by cAMP, the CREB protein attaches a transcriptional coactivator CBP (CREB- binding protein). CBP then catalyzes histone acetylation, which loosens the packing of nucleosomes and interacts with RNA polymerase to facilitate assembly of the transcription machinery at nearby gene promoters.

STAT protein (Signal Transducers and Activators of Transcription)

Interferons are the signaling molecules that activate STATs. Secreted interferons bind to receptors on the surface of neighboring cells, causing them to produce proteins that make the target cells more resistant to viral infection.

Figure show the histone acetylation by CREBs protein

Figure shows cell resistant to viral infection by STATs protein

Expression of Genes Coordinated by Heat-Shock Response Elements.

Heat-shock genes are genes that is expressed in response to an increase in

temperature. They are also called stress-response genes. The activity of Heat-shock

genes in prokaryotic and eukaryotic genomes is triggered by the environment. For

example, warming cultured cells by raising the temperature activates the transcription of

multiple heat- shock genes. The protein produced in transcription help minimize the

damage resulting from thermal denaturation of important cellular proteins.

In cells of Drosophila, the heat- shock genes Hsp70, a molecular chaperone which is

involved in normal protein folding can also facilitate the refolding of heat- damaged

proteins.

Refolding of protein by heat-shock genes

Homeotic Genes Code for Transcription Factors.

Homeotic genes code for transcription factors that regulate embryonic development.

Homeotic genes are unusual class of genes that coordinate gene regulation in

eukaryotes. Homeo means “alike”, and when mutations occur in these genes, a strange

thing happens during embryonic development – one part of the body is replaced by a

structure that is normally occurs somewhere else. For example, Drosophilla genes;

bithorax gene complex and antennapedia gene complex when mutated leads to

different developmental changes.

Bithorax Gene

-Mutation causes an additional set of wings formed in Drosophilla.

Antennapedia Gene

Mutation causes legs to develop where the antennae should be. Homeotic genes

control major developmental pathways, and in vertebrates they may also help regulate

other important processes, such as histone production and antibody synthesis.

Posttranscriptional Control

After transcription has taken place, the flow of genetic information involves a complex

series of posttranscriptional events. Posttranscriptional regulation useful in providing

ways to fine-tune the pattern of gene expression rapidly, allowing cells to respond to

transient changes in the intracellular or extracellular environment without changing their

overall transcription patterns.

Control of RNA Processing and Nuclear Export Follows Transcription .

Posttranscriptional control begins with the processing of primary RNA transcripts. All

RNA transcripts in eukaryotic nuclei undergo substantial processing :

I. Addition of a 5’ cap and a 3’ poly(A) tail.

II. Chemical modifications such as methylation, splicing together of

exons accompanied by the removal of introns and RNA editing.

RNA splicing is an important control site because its regulation allow cells to create a

variety of different mRNAs from the same pre-mRNA. This permits a gene to generate

more than one protein. This phenomenon called alternative splicing, is based on a

cell’s ability to permit some splice sites to be skipped and others to be activated. For

example , mRNA coding for antibody immunoglobulin M (IgM).

The antibody protein immunoglobulin M (IgM) exist in two forms, secreted IgM and

membrane-bound IgM. For example, viral RNAs produced in infected cells. HIV- the

virus responsible for AIDS – produces an RNA molecule that remain in the nucleus until

viral protein is synthesized.

Alternative splicing, mRNA coding for immunoglobulin M

Translation Rates Can Be Controlled by Initiation Factors and Translational Repressors.

One mRNA molecules have been exported from the nucleus to the cytoplasm, several

translational control mechanisms are available to regulate the translation of mRNA

into polypeptide. For example, translational control occurs in developing erythrocytes.

Globin polypeptide chains are the main product of translation. The synthesis of globin

depends on the availability of heme- the iron containing prosthetic group that attaches

to globin chains to form hemoglobin. So, erythrocytes have developed a mechanism for

adjusting polypeptide synthesis to match heme availability.

Translation Can Also Be Controlled by Regulation of mRNA Degradation

Translations are also subject to control by alterations in mRNA stability. Degradation

rates measured by pulse-chase experiments-cells first incubated for a period of time

( the ‘pulse’) in presence of a radioactive compound that become incorporated into

mRNA. Then ,cells are placed in a radioactive medium and incubation is continued( the

‘chase’). This methods allows researchers to measure an mRNA’s half-life.

Control of mRNA degradation in response to iron ;

RNA Interference Utilizes Small RNAs to Silence the Expression of Genes Containing Complementary Base Sequences.

Regulatory proteins like IRE-binding protein-bind to specific mRNA activity. Individual

mRNAs are also controlled by a class of a small RNA molecules that inhibit the

expressions of mRNAs containing sequences related to sequences found in small

RNAs.

When a cell encounters double-stranded RNA, Dicer cleaves it into an siRNA about 21-

22 base pair length that is subsequently combined with RISC proteins to form an

siRISC. After degradation of one of the two siRNA strands, the remaining strand bind

the siRISC via complementary base pairing to either a target mRNA molecule in

cytoplasm or to a target DNA sequence in the nucleus, thereby silencing gene

expression at either the translational or transcriptional level.

MicroRNAs Produced by Normal Cellular Genes Silence the Translation of Developmentally Important Messenger RNAs

MicroRNAs are produced by a multistep process in which a microRNA gene is first

transcribed into a primary transcript (pri-miRNA) that folds into a hairpin loop. The

nuclear enzyme Drosha xleaves pri-miRNA into a smaller(roughly 70 nucleotide) hairpin

RNA(pre-miRNA), which is exported from the nucleus and cleaved by Dicer to release a

microRNA (miRNA) molecule that is 21-22 nucleotides long. The miRNA then joins with

RISC proteins to form miRISC, which is directed by its miRNA to messenger RNAs

containing sequences complementary to those in the miRNA. Most commonly, multiple

miRISCs bind to the same messenger RNA through partly complementary sequences

and together inhibit translation. Occasionally the miRNA is exactly complementary to a

site within a particular messenger RNA, resulting in messenger RNA degradation by a

mechanism similar to that observed with siRNAs.