sGENE_EXPRESSION2

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GENE

EXPRESSION INEUKARYOTES



REGULATION OFGENE EXPRESSIONIN EUKARYOTES Regulation of eukaryotic gene

expression can occur at manylevels:

transcriptional control posttranscription control

(processing pre-mRNA) transport to the cytoplasm stability of the mRNA translational control (selecting

which mRNA are translated) posttranslational modification of

the protein product



TRANSCRIPTIONAL

CONTROL Unlike the promoters of prokaryotic cells, the eukaryoticcells have different promoter plus enhancer.

(Eukaryotes have 3 types of RNA pol and require transcription factors, TF for transcription, TF binds to enhancer and promoter)



TRANSCRIPTIONALCONTROL

Enhancer Enhancers can be on either side of a gene, at some

distance from the gene @ within the gene which canregulate a gene on any chromosome.

It interacts with multiple regulatory proteins,transcription factor and can increase the efficiencyof transcription initiation @ activate the promoter.

If an enhancer is experimentally moved to anotherlocation in the genome @ if an unrelated gene is

placed near an enhancer, the transcription of theadjacent gene is enhanced.




Enhancer (cont.) Enhancers differ from promoters. Promoters are essential for basal-level transcription,

enhancers are necessary for the full level of transcription andresponsible for the time- and tissue-specific gene expression.

Enhancers stimulate levels of transcription at a distance by: altering the configuration of the chromatin bending @ looping the DNA bring distant enhancers and

promoters into direct contact in order to form complexes withtranscription factors and polymerases.




Enhancer (cont.) Enhancer interact with multiple regulatory proteins,transcription factors and have binding sites for positive(increase transcription) & negative (decreasetranscription) gene regulators @ factors (analogous to

operators of prokaryotic cells).

These regulators @ factors also control where and whengenes are expressed.

The positive factors can be divided into 2 categories:

true activators antirepressors




Enhancer (cont.)True Activators Are modular proteins with at

least 2 functional domains: Domain to bind to DNA

sequences present in theenhancer (DNA-binding domain)

Domain to activate transcriptionvia protein-protein interaction (trans-activating domain). Thisdomain binds to RNA pol @ other

transcription factors at thepromoter.




Enhancer (cont.)Antirepressors They remodel the chromatin.

Chromatin remodeling must occur when genesare turned on @ off.

Genes in chromatin that are actively transcribedare sensitve @ even hypersensitive to in vitro digestion by DNaseI while inactive @ repressedgenes are relatively resistant to DNaseI digestion.

The principle: open chromatin is more easilydigested and more DNase sensitive than closedchromatin.



Chromatin is the complex of DNA and protein that makes up chromosomes. It is found inside

the nuclei of eukaryotic cells Nucleosomes form the fundamentalrepeating units of eukaryotic chromatin[1],which is used to pack the large eukaryoticgenomes into the nucleus

http://en.wikipedia.org/wiki/Chromosome

http://en.wikipedia.org/wiki/Cell_nucleus

http://en.wikipedia.org/wiki/Eukaryote

http://en.wikipedia.org/wiki/Cell_(biology)


http://en.wikipedia.org/wiki/Chromatin

http://en.wikipedia.org/wiki/Chromatin


http://en.wikipedia.org/wiki/Cell_(biology)


http://en.wikipedia.org/wiki/Cell_nucleus

http://en.wikipedia.org/wiki/Chromosome




Enhancer (cont.)AntirepressorsChromatin is altered by 2

different

processes:

1) Catalyzed by an ATPhydrolysis-dependentremodeling complex (eg: SWI/SNF) by: DNA-histone contacts may be

loosened. The path of the DNA around an

unaltered nucleosome coreparticle may be altered.

The conformation of thenucleosome core particle may be

altered.




Enhancer (cont.)Antirepressors2) Chromatin alteration

histone modification catalyzedby one of many histone

acetyltransferase (HAT). Whenan acetate group is added toamino acids on the histone tails, the attraction betweenthe basic histone protein andacidic DNA is lessened.

To reverse back the action,deacetylase can be targetedto remove acetate group.




Enhancer (cont.)Antirepressors

When chromatin is altered by those

2 different methods from the focal

point of an enhancer, remodeling spreadstoward the promoter of the gene and

then into the transcription unit transcription turn on.



Yeast gal Genes

gal gene – encode the enzyme forgalactose breakdown. (gal gene issimilar to lac operon)

Expression of gal genes is inducible,they are regulated by the presence@ absence of their substrate,

galactose.



Yeast gal Genes (cont.)

In the absence of galactose, the genes arenot transcribed. If the galactose is added tothe growth medium, transcription begins immediately and the mRNA concentration of the transcripts increases by a thousand fold.

However, transcription is activated only if theconcentration of glucose is low (the genesare under catabolite repression)

catabolite repression = Repression

(inactivation) of certain sugar-metabolizingoperons (eg lac) in favour of glucoseutilization when glucose is the predominantcarbon source in the environment of the cell).



Yeast gal Genes (cont.)

Gal genes: GAL1 & GAL10

The transcription of these 2 genes is controlled bya single central control region, called UASG

(upstream activating sequence) ~ 170 bp.

UASG are functionally similar to enhancers in higher

eukaryotes. The chromatin structure of the UAS isconstitutively open @ DNase hypersensitive (freeof nucleosomes).

This open chromatin structure requires theSWI/SNF remodeling complex.



Yeast gal Genes (cont.) Within UAS are 4 binding sites for the

Gal4 protein (Gal4p). These sitespermanently occupied by Gal4p,whether or not the genes have beenactivated. Gal4p is in turn negativelyregulated by Gal80p, another gal gene regulator.

Gal80p is always bound to Gal4p,

covering its activation domain. Induction occurs when the inducer, a

phosphorylated galactose binds toGal80p and/or Gal4p and alters theirstructures, exposing the Gal4pactivation domain.

These genes cannot be turned on in

the presence of glucose.

Activation occurs when there is direct contact bthe activating domain and other proteins.As a result, the transcription is activated.



DNA Methylation

Chemical modification of DNA that involvesadding @ removing methyl groups from theDNA bases.

The DNA of most eukaryotic organisms ismodified after replication by the enzyme-mediated addition of methyl groups tobases and sugars.

Usually, base methylation involves cytosine and the extent of methylation can betissue-specific.



DNA Methylation

Methylation of cytosine occurs at the 5’ position, causing themethyl group to protrude into the major groove of the DNAhelix where it can alter the binding of proteins to the DNA.

Methylation occurs most often in the cytosine of CG doublets in DNA, usually in both strands causing a marked change inthe affinity of the repressor for the operator.

Low amounts of methylatioare associated with high le

of gene expression and higlevels of methylation areassociated with low levelsgene expression



PosttranscriptionalControl Although transcriptional control is

perhaps the most obvious and widelyused mode of regulation ineukaryotes, posttranscriptional modesof regulation also occur in manyorganisms.

Posttranscriptional control involves:

1. Alternative splicing

2. mRNA stability



Posttranscriptional Control 1)Alternative splicing alternative splicing can generate different forms of a

protein, so that expression of one gene can give rise to afamily of related proteins.

Eg: The initial bovine pre-mRNA transcript is processed

into one @ two preprotachykinin mRNA (PPT mRNAs) This precursor mRNA molecule includes the genetic

information specifying 2 neuropeptides called P & K.

The P neuropeptide (alpha-PPT mRNA) is restricted totissues of the nervous system and K neuropeptide (beta-

PPT mRNA) is found predominantly in the intestine andthyroid.



Posttranscriptional Control

1)Alternative splicing (cont.) The RNA sequences for both neuropeptides are

derived from the same gene.

However, the processing of the initial RNAtranscript can occur in 2 ways: Exclusion of K exon during processing result in the

alpha-PPT mRNA which upon translation yields P

Includes both P & K exons during processing result inthe alpha and beta-PPT mRNA which upontranslation yields both P & K neuropetides

i i l



PosttranscriptionalControl

1)mRNA stability After pre-mRNA are processed and transported, theyenter the population of cytoplasmic mRNA from whichmessages are recruited for translation.

All mRNAs have their own half-life. They are degraded

some time after they are synthesized, eg: min, hours@ months are synthesized.

An mRNA includes 5’ and 3’ untranslated sequencesthat contain information for stability and address tolocate the mRNA in a particular component of the

cell. Longer-lived mRNAs can produce more protein than

short-lived ones.



PosttranscriptionalControl1)mRNA stability (cont.) There are instability elements which can be

general (for all cells) @ cell-type specific. Eg:AUUUA is found in the mRNA of oncogenes,

fos fos mRNA degraded rapidly (10-30min)

In contrast, mRNAs that code for theproduction of the dominant proteins for a

particular cell, eg: Hb mRNA in erythrocyte –half-lives more than 24 hours.



Posttranslational Control(determining protein

stability) Degradation of cellular proteins is

carried out by proteasomes thatare found in nucleus and cytosol.

Proteasomes are proteins that

consist of 4 rings of polypeptidesubunits (2 alpha subunit or caps,green in colour and 2 beta subunit,blue in colour ). It has proteolyticdigestion activity.

http://en.wikipedia.org/wiki/Image:1G0U_subunits_topview.png

http://en.wikipedia.org/wiki/Image:1G0U_subunits_sideview.png




stability) cont. Proteosomes digest proteins that have been specially

selected and marked for destruction.

Some proteins are selected because they are abnormal (misfolded @ incorrectly associated with other proteins).

Some proteins such as enzymes for glycolysis and globin of an erythrocyte have long half-life (days to weeks) butothers such as regulatory proteins to trigger cell divisionmay have a half-life of only a few minutes BUT they areall degraded by proteasomes.




stability) cont. Proteins to be degraded by proteasomes are marked

for destruction by covalent linkage to ubiquition(protein).

Ubiquitin is transferred enzymatically to a lysine

residue on the target protein. Polyubiquinated proteins are recognized by the cap of

the proteasome which removes the ubiquitin chainand unfold the target protein.

The unfolded, linear polypeptide is then threaded

through the narrow opening in the ring and passedinto the central chamber of the proteasome where it isdigested into small peptides.

The peptide products are released back into thecytosol where they are degraded into theircomponents amino acids.

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