Control of gene expression

levels of gene expression

1. chromatin level

   • histone deacetylation/ acetylation

   • histone methylation /demethyation

   • DNA methylation/ demethylation

2. transcriptional level

   • control elements + proteins

3. post-transcriptional level

   • mRNA 5’ capping + polyadenylation

   • mRNA splicing + alternative splicing

4. translational level

   • phosphorylation of translation initiation factors

   • binding of regulator proteins to 5’ UTR of mRNA

   • cytoplasmic elongation of poly A tails of mRNA

5. post-translational level

   • ubiquitination

   • proteolysis

   • biochemical modification

   • protein targeting

chromatin level: histone acetylation (up-regulate expression of genes)

• addition of (negatively charged) acetyl groups to lysine residues in histone tails which is catalysed by histone acetyltransferases

• positive charge of lysine is neutralized by addition of acetyl groups which reduces the affinity of histone tails for binding to DNA resulting in loosening of histone complex from DNA (30nm loosens into 10nm)

• this gives transcription proteins and enzymes easier access to genes, resulting in up-regulation of gene expression

chromatin level: histone deacetylation (silencing of expression)

• removal of acetyl groups from lysine residues in histone tails which is catalysed by histone deacetylases

• ionic bonds between positively charged histone tails and negatively charged DNA can form again, making DNA more compact, promoting heterochromatin formation (30nm is formed)

• this reduces the transcription of genes

chromatin level: histone methylation/ demethylation

• methylation is the addition of methyl groups to lysine or arginine amino acid residues on histone proteins catalysed by histone methyltransferases

• this can cause transcriptional activation or repression depending on position of amino acid methylated and number of methyl groups added to amino acid residue

• methylation coordinates the recruitment of chromatin-modifying enzymes which as histone acetyltransferases which can regulate chromatin condensation and nucleosome mobility to activate/ inactivate transcription

• histone demethylation is the removal of methyl groups from lysine residues on histone proteins catalysed by histone demethylases

chromatin level: DNA methylation/ demethylation

• DNA methylation is the addition of methyl groups to certain bases (usually cytosine) in DNA, catalysed by DNA methyltransferases

• this may physically impede binding of transcriptional proteins to gene or facilitate the recruitment of additional proteins such as histone deacetylases or other chromatin-remodeling proteins that can modify histones to form heterochromatin

• this reduces transcription and heavily methylated genes are not expressed

• this is essential for long-term inactivation of certain genes which occurs during normal cell differentiation

• DNA demethylation is the removal of extra methyl groups which can turn on expression of these genes, catalysed by DNA methylases

Explain the effect of DNA methylation on gene expression

• there is long-term silencing of methylated genes

• methylated DNA recruits histone deacetylases which causes the removal of acetyl group from lysine residues on histone tail, restoring positive charge to lysine residues

• ionic bonds form between positively charged histone tails and negatively charged DNA, leading to compaction of chromatin

• thus, transcription factors and RNA polymerase cannot access the promoter of genes

Explain why pattern of DNA methylation differs in different cell types

• all somatic cells contain the same set of genes

• cells undergo differential gene expression

• cell-type specific genes are only expressed in that specific cell hence are not methylated

Why is this certain gene not methylated

• the gene could be a housekeeping gene that is required for normal functioning of all cell types

• for example, it could be a gene that codes for RNA polymerase

transcriptional level: activator proteins and enhancer (increase)

• DNA bending protein bends DNA

• Activator proteins via its DNA binding domain binds to enhancer

• Activator proteins then bonds via activation domain to co-activators, which in turn bind to general transcription factors and RNA polymerase II via protein-protein interactions

• this facilitates the efficient positioning of RNA polymerase II to promoter as it prevents premature disassociation of RNA polymerase II

• thus, increasing the rate of transcription

transcriptional level: repressor proteins and silencer (decrease)

• repressor binds to silencer to turn off or reduce transcription even in the presence of activator proteins

  • By blocking binding of activator proteins to enhancer when silencer is located close to enhancer thus reducing transcription

  • by blocking assembly of components of transcription initiation complex at the promoter when silencer is near promoter thus turning off transcription

  • repressor binds to silencer which is within enhancer and acts to reduce transcription by preventing activator from binding to enhancer

suggest how transcriptional repressors can interfere with action of transcriptional activators

1. repressor binds to silencer sequence which is within or overlapping an enhancer. This blocks the binding of activator to enhancer sequence thus preventing efficient positioning of RNA polymerase on promoter

2. repressor binds to an activator and changed the 3D conformation of activator’s activation domain. This prevents the binding of activator to co-activators via activation domain, thus preventing efficient positioning of RNA polymerase on promoter

3. repressor binds to activation and changed the 3D conformation of DNA-binding domain. This prevents the binding of activator to enhancer sequence via its DNA-binding domain, thus preventing efficient positioning of RNA polymerase on promoter

advantage and disadvantage of transcriptional level control

advantage:

• saves resources as there is no need to perform transcription, translation unless needed

disadvantage:

• time is needed to see the effects of transcriptional level modification

post-transcriptional modification order

1. 5’ capping

2. splicing

3. 3’ polyadenylation

post-transcriptional level: 5’ capping

• addition of modified methylated guanine nucleotide to the first nucleotide of pre-mRNA at 5’ end via a 5’-5’ triphosphate bridge

• this process is catalysed by capping enzymes like guanyltransferase

• cap-binding protein(s) associate with the 5’ cap which increases mRNA stability by protecting mRNA from degradation by 5’ exonucleases and facilitates the export of mRNA from nucleus to cytoplasm as the cap-binding proteins complex is recognised by nuclear pore complex

post-transcriptional level: splicing

• process whereby introns are excised and exons are ligated which is carried out by a complex of proteins known as spliceosomes

• this ensures mRNA sequence is continuous for translation by ribosomes

steps:

1. spliceosomes recognises splice sites and fold pre-mRNA into correct orientation for splicing and catalyses the excision of introns and ligation of exons

2. spliceosomes cleave at 5’ end of introns

3. the cleaved end joins the branch point sequence to form a lariat

4. 3’ end of intron is then cleaved

5. excision of introns

6. ligation of exons

7. spliceosomes dissociate once splicing is completed

post-transcriptional level: alternative splicing (only for some pre-mRNA)

• for same gene and pre-mRNA transcript, alternative splicing can occur to allow for the production of two or more mature mRnA transcripts

• this allows a single gene to encode for different polypeptides

• biological advantage: two or more polypeptide sequences can be derived from a single gene which allows organisms to carry fewer genes in their genome

translational level: cytoplasmic elongation of poly A tails of mRNA

• it is possible to elongate poly (A) tails again as a signal for initiation of translation

• this occurs in the cytoplasm

• this affects the stability of mRNA in cytoplasm which can lead to higher levels of protein expression

translational level: Phosphorylation of translation initiation factors

• translation initiation factors are required to begin protein translation

• phosphorylation of different initiation factors can inhibit or increase translation

• regulation of translation by reversible phosphorylation is faster than synthesis or degradation of translation initiation factors

translational level: binding of regulatory proteins to 5’ UTR of mRNA

• these regulatory proteins inhibit translation by preventing ribosomal attachment

example: synthesis of ferritin (iron-storage protein)

• its regulation is achieved through binding of translation repressor protein to 5’ UTR of ferritin mRNA, thus blocking translation of mRNA when iron concentration is low

• if concentration of iron in cytosol rises, iron binds to translation repressor protein, releasing the protein from 5’ UTR of ferritin mRNA and unblock translation, resulting in rapid increase of production of ferritin

post-translational level: protein targeting

• cells can indirectly alter gene expression levels by altering the rate at which proteins are targeted to their destinations in the cell

• this is because proteins must be transported to its specific intracellular location or exported from the cell to assume its function

• transportation of proteins to target destinations in the cell is mediated by signal sequences at N-terminus of some proteins

• once transported, the signal sequences is enzymatically removed

post-translational level: proteolysis

• the hydrolytic processing of eukaryotic polypeptides to yield smaller functional proteins

post-translational level: biochemical modifications

covalent addition of one or more groups to amino acids in a particular protein to make it functional

1. glycosylation to yield functional glycoproteins

   • short oligosaccharide proteins are added to yield glycoproteins for cell-cell recognition and adhesion

2. phosphorylation to increase or decrease function

   • regulatory proteins can be activated or inactivated by reversible addition of phosphate groups to serine, tyrosine or theronine residues on polypeptides which is catalysed by kinase enzyme

3. acetylation to increase or decrease function

   • addition of acetyl groups to histone tails result in nucleosomes being unable to bind to other nucleosomes, resulting in loosening to 30nm to 10nm chromatin fibre

post-translational level: protein degradation via ubiquitination

• proteins are marked for destruction by attaching molecules of ubiquitin

• the covalent attachment of ubiquitin molecules to protein marks it for degradation by proteasome

• the proteasomes are found in nucleus and cytoplasm of eukaryotic cells and degrade proteins by proteolysis which results in breakage of peptide bonds

• this is so that proteins do not remain too long in the cytoplasm or are active when not needed