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To know and explain:

Regulation of Bacterial Gene Expression

Constitutive ( house keeping) vs. Controllable genes

OPERON structure and its role in gene regulation

Regulation of Eukaryotic Gene Expression at different levels:

DNA methylation

Histone modifications (Chromatin Remodeling)

Increasing the number of gene copies (gene amplification)

Changing the rate of initiation of transcription

Alternate splicing , mRNA stability

Changing the rate of initiation of translation

Introduction Gene expression is the combined process

of :

othe transcription of a gene into mRNA,

othe processing of that mRNA, and

oits translation into protein (for protein-encoding genes).

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Genes are subunits of DNA, the information database of a cell that is contained inside the cell nucleus.

This DNA carries the genetic blueprint that is used to make all the proteins the cell needs.

Every gene contains a particular set of instructions that code for a specific protein

Genes can be classified into two types according to their expression:

1. Constitutive genes:

They are expressed permanently at a fixed rate, irrespective of the cell condition and characterized by simple structures (housekeeping genes).

2. Inducible genes:

They are expressed temporarily only as needed by the cell, so that their amount may increase or decrease under different conditions and their structure are more complicated. The regulation of expression usually involves this group of genes.

Gene Expression We can say that Gene “A” is being expressed if:

Gene “A” is being transcribed to form mRNA

That mRNA is being translated to form proteins

Those proteins are properly folded and are in state where they can be used by the cell

Cells are extremely selective about what genes are expressed, in what amounts, and when ?

Gene expression can be regulated at a variety of different stages.

Levels of regulation of gene expression

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When the expression of genetic information is quantitatively increased by the presence of specific regulatory element is known as positive regulation.

Element modulating positive regulation is known as activator or positive regulator.

When the expression of genetic information is diminished by

the presence of specific regulatory element is known as

negative regulation.

The element or molecule mediating the negative regulation is

said to be repressor.

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Type A response:

Type B response:

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1) RNA polymerase binds to DNA at promoters.

2) Transcription initiation is regulated by proteins

that bind to or near promoters.

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Synthesis of the primary RNA transcript (transcription)

Posttranscriptional modification of mRNA

Messenger RNA degradation

Protein synthesis ( translation )

Posttranslational modification of proteins

Protein targeting & transport

Protein degradation 13

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Regulation of Gene Expression

Gene expression can be regulated: During transcription (transcriptional control).

During translation (translational control).

After translation (post-translational control).

Some Examples of Regulation of Gene Expression

Transcriptional control: Regulatory proteins affect the ability for RNA polymerase

to bind to or transcribe a particular gene.

Translational control: Various proteins may affect rate of translation.

Enzymes may affect lifetime of a mRNA transcript.

Post-translational control: Translated protein may be modified by phosphorylation,

which may change its folding and/or activity.

In genetics, an operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single promoter and regulated by a common operator.

Operons occur primarily in prokaryotes but also in some eukaryotes.

It is defined as a set of adjacent structural genes, plus the adjacent regulatory signals that affect transcription of the structural genes.

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An operon is made up of 4 basic DNA components:

Promoter – a nucleotide sequence that enables a gene to be transcribed. The promoter is recognized by RNA polymerase, which is then initiates transcription.

Regulator – These genes control the operator gene in cooperation with certain compounds called inducers and corepressors present in the cytoplasm.

Operator – a segment of DNA that a repressor binds to. It is classically defined in the lac operon as a segment between the promoter and the genes of the operon.

Structural genes – the genes that are co-regulated by the operon.

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Operon regulation can be either negative or positive by induction or repression.

In negative inducible operons, a regulatory repressor protein is normally bound to the operator, which prevents the transcription of the genes on the operon .

If an inducer molecule is present, it binds to the repressor and changes its conformation so that it is unable to bind to the operator. This allows for expression of the operon.

The lac operon is a negatively controlled inducible operon, where the

inducer molecule is allolactose.

In negative repressible operons, transcription of the operon normally takes place.

The trp operon, involved in the synthesis of tryptophan (which itself

acts as the corepressor ), is a negatively controlled repressible operon.

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With positive control, an activator protein stimulates transcription by binding to DNA.

In positive inducible operons, the activator proteins are normally unable to bind to the relevantDNA.

When an inducer is bound by the activator protein, it undergoes a change in conformation so that it can bind to the DNA and activate transcription.

In positive repressible operons ( suppress), the activator proteins are normally bound to the pertinent DNA segment.

However, when an inhibitor is bound by the activator, it is prevented from binding the DNA.

This stops activation and transcription of the system.

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Regulation of gene expression in prokaryotes

Bacteria are programmed to grow fast, but not all proteins that a cell can make are needed all the time .

The mRNA for most inducible proteins have very high turnover with a half-life of less than 2 minutes. Therefore, the primary function of transcriptional gene control is to adjust cells need of proteins according to changing

environmental conditions.

Lactose (milk sugar) is a disaccharide that can be used as energy source by

E coli when the enzyme β-galactosidase catabolizes it to glucose and galactose monosaccharides .

Normally bacteria prefer glucose as energy source; therefore if glucose is present in the

medium and lactose is missing the

catabolism of lactose is suppressed.

However, when glucose is absent and lactose is present in the medium the gene for

β-galactosidase enzyme (lac Z gene )

becomes induced.

This enzyme induction is initiated by control system of lactose catabolism called Lac operon

which also induces two other enzymes β-

galactoside permease (coded for by the lacY

gene) and β -galactoside acetyltransferase

(coded for by lacA gene) .

The permease is involved in transport of lactose across the membrane while the transferase protects other intracellular galactosides from the activity of β-galactosidase.

All 3 structural genes are located next to each other on the chromosome and also expressed together during the catabolism of lactose .This expression results in the formation of a single polycistronic (polygenic) mRNA that will be subdivided into 3 segments, each will produce a single enzyme.

Polycistronic A single mRNA encoding several different

polypeptide chains.

Cistron A section of DNA that contains the

genetic code for a single polypeptide and functions as a hereditary unit.

The polycistronic mRNA is synthesized by RNA polymerase that binds at a single promoter.

located near to the lac Z gene.

A fourth gene, the regulator gene ( lac I) is located adjacent to the other three genes but its product the lac repressor is constitutively expressed by different RNA polymerase that binds a separate promoter.

In the absence of lactose, the repressor is bound to a regulatory region called the operator located near to the promoter but this binding interferes with the attachment of sigma factor to RNA polymerase and therefore prevents the expression of all three enzymes.

This type of transcriptional control in which structural genes are initially present in suppressed state is called negative regulation of gene expression. Also, the operon is considered a sequence of genes present under the transcriptional control of the same operator.

The mode of action of the lac repressor

Gene Expression Some genes are constitutively expressed:

Expressed equally at all times.

Many other genes are regulated and their expression may be induced or repressed: Gene expression is not just “on” or “off” - it can vary from

one condition to another

Lactose Metabolism in E. coli

Bacteria break down lactose into its component monomers, glucose and galactose, using the enzyme -galactosidase.

Bacteria can then use the monomers to generate ATP through cellular respiration.

Lactose Metabolism in E. coli

Lactose gets transported (imported) into the cell with the help of galactoside permease.

Once inside the cell, lactose can be cleaved into monomers with the help of -galactosidase.

Lactose Metabolism in E. coli The expression of the gene for -galactosidase

(and galactoside permease) is regulated by lactose and glucose:

High levels of LACTOSE stimulate (induce) the

expression of the gene for -galactosidase.

High levels of GLUCOSE inhibit (repress) the expression of the gene for -galactosidase.

How (by what mechanisms) do lactose and glucose regulate gene expression in E. coli ?

Lactose Metabolism Mutants Monod and Jacob in 1961 conducted a

genetic screen for E. coli mutants that were specifically defective in lactose metabolism:

Generated a large number of E. coli

colonies bearing mutations in random locations in their genomes.

“screened” through these colonies to find ones that could not successfully grow on a “lactose only” medium, but could grow on a “glucose only” medium.

Screen Results

Three classes of E. coli mutants defective in lactose metabolism were found:

1.) lacZ- mutants lacked functional -galactosidase.

2.) lacY- mutants lacked the membrane protein galactosidase permease.

Screen Results 3.) lac I- mutants:

Produce -galactosidase and galactoside permease even when lactose is absent.

lacI- mutants are constitutive mutants:

They produce product(s) at all times.

Loss of regulation by lactose.

The lacI gene must code for a protein that normally serves to repress expression of the lacZ and lacY genes when lactose is absent.

Screen Conclusions

The lacZ gene codes for: -galactosidase.

The lacY gene codes for: Galactoside permease.

The lacI gene codes for: A regulatory protein (a repressor) that normally

functions to repress lacZ and lacY gene expression when lactose is absent.

The lac Genes

The lacZ, lacY, and lacI genes are in close physical proximity to each other on the bacterial chromosome.

How does the lacI gene product repress gene expression of lacZ and lacY?

Negative Control of Transcription

Negative control occurs when:

A regulatory protein (a repressor) binds to DNA and decreases the rate of transcription of downstream genes.

The lacI gene codes for a repressor, a regulatory protein that exerts negative control over the lacZ and lacY genes.

Positive Control of Transcription

Positive control occurs when:

A regulatory protein (an activator) binds to DNA and RNA polymerase the rate of transcription of downstream genes increased.

Negative Control of lacZ and lacY Gene Expression

The lacI gene codes for a repressor that binds to DNA just downstream of the promoter, physically blocking transcription of lacZ and lacY.

Negative Control of lacZ and lacY Gene Expression

In the presence of lactose: Lactose binds to the repressor.

Lactose-repressor complex releases from DNA.

RNA polymerase can now transcribe lacZ and lacY.

Thus, lactose induces transcription by preventing the repressor from exerting negative control.

Negative Control of lacZ and lacY Gene

Expression

If the lacI gene is mutated (lacI- mutants):

No functional repressor is synthesized.

No repressor is ever present in the cell to bind downstream of the promoter to block transcription.

lacZ and lacY will be transcribed regardless of whether lactose is present or absent.

Summary

Positive Control of the lac Operon

Binding of CAP (an activator protein) to the CAP site (a DNAregion) just upstream of the lac operon promoter greatly increases lac operon gene expression.

CAP can only bind the CAP site if CAP is bound to cAMP (cyclic AMP).

Positive Control of the lac Operon

If cAMP levels are low, CAP is inactive and won’t bind to the CAP site, and therefore transcription will be infrequent.

What regulates cAMP levels?

Glucose Regulates cAMP Levels

cAMP is synthesized from ATP by an enzyme called adenylyl cyclase.

Glucose inhibits adenylyl cyclase activity.

If glucose levels are high, this will cause cAMP levels in the cell to be low.

Glucose Regulates cAMP Levels

In this way, glucose, by regulating cAMP levels, can regulate the expression of the lac operon genes.

Another Effect of Glucose

Recent studies have revealed a second, perhaps even more significant, contribution of glucose to regulating lac operon expression:

High level of glucose leads to a dephosphorylation and inactivation of gal. permease in E. coli cell membrane…

…which inhibits lactose transport into the cell…

….which prevents lactose accumulation inside the cell…

…which allows the lacI repressor protein to bind to the lac operator, decreasing lac operon gene expression.

Summary: The lac Operon

END Part I

Biochemistry for medics-Lecture notes 55