GENE EXPRESSION IN EUKARYOTES

REGULATION OF GENE EXPRESSION IN EUKARYOTES

Regulation of eukaryotic gene expression can occur at many levels:

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 eukaryotic cells 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)

TRANSCRIPTIONAL CONTROL Enhancer

Enhancers can be on either side of a gene, at some distance from the gene @ within the gene which can regulate a gene on any chromosome. It interacts with multiple regulatory proteins, transcription factor and can increase the efficiency of transcription initiation @ activate the promoter. If an enhancer is experimentally moved to another location in the genome @ if an unrelated gene is placed near an enhancer, the transcription of the adjacent gene is enhanced.

TRANSCRIPTIONAL CONTROL Enhancer (cont.) Enhancers differ from promoters.

Promoters are essential for basal-level transcription, enhancers are necessary for the full level of transcription and responsible 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 with transcription factors and polymerases.

TRANSCRIPTIONAL CONTROL Enhancer (cont.)

Enhancer interact with multiple regulatory proteins, transcription factors and have binding sites for positive (increase transcription) & negative (decrease transcription) gene regulators @ factors (analogous to operators of prokaryotic cells). These regulators @ factors also control where and when genes are expressed. The positive factors can be divided into 2 categories:
true

activators antirepressors

TRANSCRIPTIONAL CONTROL Enhancer (cont.)

True Activators

Domain

Are modular proteins with at least 2 functional domains:

to bind to DNA sequences present in the enhancer (DNA-binding domain) Domain to activate transcription via protein-protein interaction (trans-activating domain). This domain binds to RNA pol @ other transcription factors at the promoter.

TRANSCRIPTIONAL CONTROL Enhancer (cont.)

Antirepressors

They remodel the chromatin. Chromatin remodeling must occur when genes are turned on @ off. Genes in chromatin that are actively transcribed are sensitve @ even hypersensitive to in vitro digestion by DNaseI while inactive @ repressed genes are relatively resistant to DNaseI digestion. The principle: open chromatin is more easily digested and more DNase sensitive than closed chromatin.

Chromatin is the complex of DNA and protein that makes up chromosomes. It is found inside the nuclei of eukaryotic cells Nucleosomes form the fundamental repeating units of eukaryotic chromatin[1], which is used to pack the large eukaryotic genomes into the nucleus

TRANSCRIPTIONAL CONTROL Enhancer (cont.)

Antirepressors
Chromatin is altered by 2 different processes: 1) Catalyzed by an ATP hydrolysis-dependent remodeling complex (eg: SWI/ SNF) by:

DNA-histone contacts may be loosened. The path of the DNA around an unaltered nucleosome core particle may be altered. The conformation of the nucleosome core particle may be altered.

TRANSCRIPTIONAL CONTROL Enhancer (cont.) Antirepressors

2) Chromatin alteration histone modification catalyzed by one of many histone acetyltransferase (HAT). When an acetate group is added to amino acids on the histone tails, the attraction between the basic histone protein and acidic DNA is lessened. To reverse back the action, deacetylase can be targeted to remove acetate group.

TRANSCRIPTIONAL CONTROL Enhancer (cont.)

Antirepressors When chromatin is altered by those 2 different methods from the focal
point of an enhancer, remodeling spreads toward the promoter of the gene and then into the transcription unit transcription turn on.

Yeast gal Genes

gal gene encode the enzyme for galactose breakdown. (gal gene is similar 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 are not transcribed. If the galactose is added to the growth medium, transcription begins immediately and the mRNA concentration of the transcripts increases by a thousand fold. However, transcription is activated only if the concentration of glucose is low (the genes are under catabolite repression) catabolite repression = Repression (inactivation) of certain sugar-metabolizing operons (eg lac) in favour of glucose utilization when glucose is the predominant carbon source in the environment of the cell).

Yeast gal Genes (cont.)

Gal genes: GAL1 & GAL10 The transcription of these 2 genes is controlled by a 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 is constitutively open @ DNase hypersensitive (free of nucleosomes). This open chromatin structure requires the SWI/SNF remodeling complex.

Yeast gal Genes (cont.)

Within UAS are 4 binding sites for the Gal4 protein (Gal4p). These sites permanently occupied by Gal4p, whether or not the genes have been activated. Gal4p is in turn negatively regulated 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 to Gal80p and/or Gal4p and alters their structures, exposing the Gal4p activation domain. These genes cannot be turned on in the presence of glucose.

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

DNA Methylation
Chemical modification of DNA that involves adding @ removing methyl groups from the DNA bases. The DNA of most eukaryotic organisms is modified after replication by the enzymemediated addition of methyl groups to bases and sugars. Usually, base methylation involves cytosine and the extent of methylation can be tissue-specific.

DNA Methylation

Methylation of cytosine occurs at the 5 position, causing the methyl group to protrude into the major groove of the DNA helix 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 in the affinity of the repressor for the operator.

Low amounts of methylation are associated with high lev of gene expression and high levels of methylation are associated with low levels o gene expression

Posttranscriptional Control

1. 2.

Although transcriptional control is perhaps the most obvious and widely used mode of regulation in eukaryotes, posttranscriptional modes of regulation also occur in many organisms. Posttranscriptional control involves: Alternative splicing 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 a family 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 to tissues of the nervous system and K neuropeptide (betaPPT mRNA) is found predominantly in the intestine and thyroid.

Posttranscriptional Control
1)Alternative splicing (cont.)

The RNA sequences for both neuropeptides are derived from the same gene. However, the processing of the initial RNA transcript 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 in the alpha and beta-PPT mRNA which upon translation yields both P & K neuropetides

Posttranscriptional Control
1)mRNA stability

After pre-mRNA are processed and transported, they enter the population of cytoplasmic mRNA from which messages 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 sequences that contain information for stability and address to locate the mRNA in a particular component of the cell. Longer-lived mRNAs can produce more protein than short-lived ones.

Posttranscriptional Control
1)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 (10-30 min) In contrast, mRNAs that code for the production 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 that are found in nucleus and cytosol. Proteasomes are proteins that consist of 4 rings of polypeptide subunits (2 alpha subunit or caps, green in colour and 2 beta subunit, blue in colour). It has proteolytic digestion activity.

Posttranslational Control (determining protein 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) but others such as regulatory proteins to trigger cell division may have a half-life of only a few minutes BUT they are all degraded by proteasomes.

Posttranslational Control (determining protein 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 chain and unfold the target protein. The unfolded, linear polypeptide is then threaded through the narrow opening in the ring and passed into the central chamber of the proteasome where it is digested into small peptides. The peptide products are released back into the cytosol where they are degraded into their components amino acids.