Chapter 2: Gene Expression and Epigenetics Molecular Diagnostics Lela Buckingham

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What is transcription

A way of gene expression. Done by copying 1 strand of DNA into RNA via RNA polymerase. Cells with the same genotype can have vastly different phenotypes due to structural changes in DNA from methylation

Which stage of the cell cycle does transcription/translation occur

interphase

RNA polymerase I and III codes for

noncoding RNA

RNA polymerase II codes for

mRNA

RNA Polymerase: E coli RNA Polymerase II

Uses DNA to produce mRNA

RNA Polymerase: RNA Poly I

Uses DNA to produce rRNA

RNA Polymerase: RNA Poly II

Uses DNA to produce mRNA

RNA Polymerase: RNA Poly III

Uses DNA to produce tRNA and snRNA

RNA Polymerase: mitoRNA polymerase

Uses DNA to produce mRNA in the mitochrondria

RNA Polymerase :mammalian DNA poly a

Uses DNA to produce primers

RNA Polymerase: Dengue Virus Polymerase and HCV RNA Polymerase

Uses RNA to produce its own viral genome

RNA Polymerase: PolyA polymerase

Produces PolyA tails without a template

How is information in DNA utilized

It has be to transcribed and then translated into protein

Cellular activity and location of RNA Polymerase I in eukaryotes

Found in the nucleolus, Produces 18S, 58S, and 28S rRNA insensitive to alpha-amanitin

Cellular activity and location of RNA Polymerase II in eukaryotes

Found in the nucleus. Produces mRNA and snRNA and Inhibited by alpha-amanitin

Cellular activity and location of RNA Polymerase III in eukaryotes

Found in the nucleus. Produces tRNA and 5s rRNA. Inhibited by high concentration of alpha-amanitin

Transcription initiation

RNA and its accessory proteins assemble at start site at DNA (Promoter). RNA has a lot more transcription promoter sites when compared to DNA even though DNA and RNA catalyze basically the same reaction. Also, RNA polymerases work with different proteins to find/bind DNA for transcription preparation.

Transcription elongation

RNA is synthesized using the base sequence of one strand of the double helix (anti-sense strand). RNA Polymerase move slower than DNA polymerase but DON'T REQUIRE PRIMING. 1st ribonucleoside triphosphate retains all of the phosphate groups but the subsequent ones only retain alpha PO4. 5'5' covalent cap attached to methylated guanine residue after initiation of RNA synthesis. The Cap protects the end of the growing pre-mRNA, facilitates ribosome binding and efficient translation

Transcription Termination In prokaryotes

increase in RNA synthesis occurs when protein products are low, increase levels of gene product induce termination of synthesis. Done by interaction of RNA polymerase w/ DNA template or Rho interaction. Rho interaction starts at the G-C rich area hairpin and stops at the A-T rich area.

RNA Polymerase I Termination factors

Sal box and TTF 1

RNA Polymerase II Termination factors

Alternate polyadylation sites

RNA Polymerase III Termination factors

adenine residues/TF

Transcription Termination In eukaryotes

mRNA synthesis (catalyzed by Pol II) Proceeds until it reaches a polyA site. The just formed mRNA is released by an endonuclease and subsequent RNA is degraded 5' to 3' towards RNA polymerase until it reaches PolyA site where transcription stops and PolyAdenate Polymerase adds PolyA tail of 20-200 Adenine. This is important for stability, localization, and translation

lac operon

a gene system whose operator gene and three structural genes control lactose metabolism in E. coli

Regulation of Transcription

Gene expression is a key determinant of phenotype. Can be constant or have tight regulation. Stoichiometric balance with specific combinations of proteins are crucial for all differentiation/development protein availability; Protein availability and function are controlled via transcription, translation, and protein modification

constitutive

always on, continual use within the cell

Regulation of mRNA synthesis at initiation is done by

Cis factors and Trans factors

cis factors

DNA sequences that mark places on DNA involved in initiation and control of RNA synthesis

Trans factors

distant, diffusible, proteins (activators and repressors) that interact with the cis elements

operon

A unit of genetic function common in bacteria and phages, consisting of coordinately regulated clusters of genes with related functions and small genomes. Transcribed on one mRNA and subsequently separated into individual proteins

Operons are controlled by binding regulatory trans factors to cis sequences preceding the structural genes

Enzyme Repression

type of negative control where a co-repressor must bind to a repressor in order to turn off transcription

enzyme induction

inducer will bind to the repressor to leave the operator free to turn on expression for transcription

Enzyme attenuation

regulation through formation of stems and loops in the RNA transcript by intra-strand H bonding of complementary bases

Regulates expression at the translational level based on amount of enzyme present

Anti-termination- will proceed with translation

Termination- high levels will get stopped by hairpin to stop translation

cis regulatory elements: Prokaryotes

Their regulatory elements are close to the structural gene they control near the promoter

Cis regulatory elements: Eukaryotes

Distal elements can be far away from the genes they control, proximal elements cam be located in or around the genes. Elements can be behind the genes. more complex.

If Eukaryotes don't have operons, how are genes expressed

Synchronous expression via combination control. Similar patterns cause similar cis elements to respond simultaneously to specific combinations of controlling trans factors

post-transcriptional regulation: Prokaryotes

only changes through transcription process can cause changes in gene expression and transcription and translation is concurrent. RNA stability can be affected by secondary structure and polyadenylation

Post-transcriptional regulation: Eukaryotes

Trickier; Regulated by RNA processing steps and Alternate splicing. Alternate splicing with protein factors are responsible for tissue and development specific gene expression patterns

Circular RNA

Controls gene expression. Endogenous byproducts of RNA splicing in protein coded genes. Roles are: protein binding, activation of transcription, sub-cellular localization, stable. Implicated int tissue development, stress response, and cancer

Post Translational regulation

Peptides and proteins are modified with lipids, sugars, and other factors by cellular enzymes such as kinases, phosphatases, ubiquitinases, transferases, acetylases, methylases, and ligases

Gene expression is measured in the lab at the level of transcription and protein production

Epigenetics

Phenomenon that allows cells to take on different phenotypes without changing its genotype. Changes occur at the meiotic and mitotic level not encoded in genotype. Rapid and heritable due to environmental changes w/o changing DNA. Modified by chromatin and noncoding RNA

histone modification

adding chemical modifications to proteins called histones that are involved in packaging DNA. Chromatin nuclear DNA and associated proteins, compacted into nucleosomes in eukaryotes

Nucleosome

150 bp of DNA wrapped around 8 histone proteins: 2 Histone 2A, 2B, Histone 3, Histone 4. This modifies histone by modifying chromatin protein and transcription factors affecting gene production.

Occurs on specific amino acid and carboxy ends of histone protein sequences

What are ways that nucleosomes can modify histones to affect gene production

Methylation, phosphorylation, ubiquination, and acetylation.

How to notate the Histone Code

Nomenclature for Core Histone + Amino Acid Position (single letter code) + Modification '

Acylation of lysine at position 27 @ Histone H3= H3K27Ac

Histone H2A Acetylation Function

activation

Histone H2A phosphorylation function

mitosis/DNA repair Function depends on amino acid modified or combination of modifications present

Histone H2A Ubiqutylation function

stem cells

Histone H2B Acetylation Function

activation (Like H2A)

Histone H2B phosphorylation function

Apoptosis

Histone H2B Ubiqutylation function

Transcription

Histone H3 unmodified function

silencing

Histone H3 methylation function

silencing/activation Function depends on amino acid modified or combination of modifications present

Histone H3 Phosphorylation function

mitosis/activation Function depends on amino acid modified or combination of modifications present

H3 Histone Acetylation Function

Activation/histone positioning Function depends on amino acid modified or combination of modifications present

Histone H4 phosphorylation function

Activation

Histone H4 methylation function

silencing and activation Function depends on amino acid modified or combination of modifications present

H4 Histone Acetylation Function

DNA repair and histone positioning

What are histone deactylase inhibitors used for

hematological malignancies (cancers) and can be used as a therapeutic or diagnostic tool

Non-Polar Amino Acids

Alanine, Leucine, Isoleucine, Proline, Phenylalanine, Methionine, Tryptophan, Valine

polar amino acids

Serine, Threonine, Tyrosine, Asparagine, Cysteine, Glutamine, Glycine, Proline

Negatively Charged amino acids

aspartic acid, glutamic acid

Positively charged amino acids

lysine, arginine, histidine

alanine abbreviation and letter

Ala, A

Isoleucine abbreviation and letter

Ile, I

Leucine abbreviation and letter

Leu, L

Metionine abbreviation and letter

Met, M

Phenylalanine abbreviation and letter

Phe, F

Tryptophan abbreviation and letter

Trp, W

Valine abbreviation and letter

val, V

Asparagine abbreviation and letter

Asn, N

Cysteine abbreviation and letter

Cys, C

Glutamine abbreviation and letter

Gln, Q

Glycine abbreviation and letter

Gly, G

Proline abbreviation and letter

Pro, P

Serine abbreviation and letter

Ser, S

Threonine abbreviation and letter

Thr, T

Tyrosine abbreviation and letter

Tyr, Y

Aspartic Acid abbreviation and letter

Asp, D

Glutamic Acid abbreviation and letter

Glu, E

Arginine abbreviation and letter

Arg, R

Histidine abbreviation and letter

His, H

Lysine abbreviation and letter

Lys, K

DNA methylation is done with ________________ in prokaryotes/eukaryotes

epigenetic regulation

Where does epigenetic regulation occur in vertebrates

In CG rich areas or regions of more than 200 bp in length with an observed/expected ration of greater than 0.6. Found around: 1st exons, promoter regions and 3' end of genes

Imprinting

mainly done in the methylation of DNA stage. Gamete specific silencing of genes. Maintains balanced expression of genes in growth and embryonic development by selective methylation of homogenous genes. Examples where imprinting made a difference: mules vs hinnies, Angelmann vs Prader Willi Syndrome. This phenomenon may also be partially responsible for abnormal development of phenotypic characteristics of cloned animals because cloning bypasses gametogenesis

Mules and hinnies are both the resulting of hybridizing between a horse and a donkey. This shows the difference between

imprinting during oogenesis and spermatogenesis

Angelmann/Prader Willi

show genomic imprinting problems on chromsome 15

What are the four enzymes that methylate DNA

DNMT 1- maintenance enzyme for hemi-methylated DNA

DNMT 3a and 3b- de novo methylase that methylates un methylated DNA establishing newly methylated regions as seen in imprinting

DNMTL- non functional. May regulate 3a/3b by occupying DNA and protein sites.

DNMT

DNA methyltransferase

DNA methyltransferase 2 may modify

bases in tRNA and others

DNMT 1 and 3 may recognize

cytosine followed by guanine bases based on their symmetry. These enzymes are recruited to methylated areas by proteins that bind histones and hemi-methylated DNA

Transcriptional silencing

responsible for X chromosome silencing in females and position effects (Silencing genes when placed in heterochromic areas)

CpG islands

DNA regions rich in C residues adjacent to G residues. Especially abundant in promoters, these regions are where methylation of cytosine usually occurs.

Normal CpG Islands

unmethylated or transiently methylated islands before active genes. Areas can also have heavier methylation before coding sequences

Abnormal in CpG islands

Colon and Brain cancer causing hypermethylation CpG island Methylator Phenotype (CIMP), Hypomethylation can also occur in tumor cells and if it occurs around long interspersed nuclear elements (LINES) it can cause genomic instability in cancers and chromosome alterations/breakages

DNA demethylation

The removal of the methyl group from methylated DNA enabling genes to become active so they can be transcribed

Ten-Eleven Translocation Enzymes (TETs)

enzyme which catalyzes demethylation through a series of oxidative reduction reactions. Major pathway of active demethylation in vertebrates and require a-ketogluterate to function

1.) 5-methylcytosine-->hydroxymethylcytosine

2.)hydroxymethylcytosine -> 5 formyl methyl cytosine

3.) 5 formyl methyl cytosine-> 5 carboxymethyl cytosine

4. Decarboxylase enzyme is added to restore 5 methyl cytosine

RNA methylation

occurs in tRNA, rRNA, mRNA, and ncRNA. Usually on N6-methyladenosine @ GAC or AAC. 25% have m6a around stop codons. Sometimes found in 5' untranslated regions of RNA.

This effects RNA stability, splicing, and translation

Methyltransferase enzymes RNA

METTL14 and METTL13