Genetics Exam 3

Types of viral genomes

• DNA/RNA viruses: single or double stranded

• RNA ←→ DNA viruses:

  • retroviruses: ssRNA → DSDNA

  • hepaDNAviruses: partially dsDNA

*are diverse: diff sizes, linearity, size, etc.

Supercoiling

coils in DNA that occur during replication and transcription

• facilitations compacting of DNA into bacterial chromosomes

• done by topoisomerases

  • enzymes that cut 1/both DNA strands

  • winds/unwinds helix before resealing ends

  • found in both prokaryotes and eukaryotes

Polytene chromosomes vs lampbrush

giant chromosomes that form due to repeated replication w/o division

• puff regions: areas of unwound chromatin

  • due to high activity/high gene expression

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type of huge chromosome found in vertebrate oocytes (immature eggs) and some spermatocytes of insects

contain large amounts of looping and uncondensing

occur as DNA is actively being transcribed (into RNA)

can be isolated/removed to be studied during diplotene stage

of prophase I

Chromatin, Chromatin remodeling

decondensed, uncoiled chromosomes in interphase…

Euchromatin

• Uncoiled and active

• Appears unstained during interphase

Heterochromatin

• Condensed areas are mostly inactive

• Appears stained during interphase

*chromosomal banding techniques: Differential staining along longitudinal axis of mitotic chromosome (Staining patterns resemble polytene chromosome bands)

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Remodeling:

• structure changes to accommodate DNA interactions

  • relax the compact structure

  • expose DNA regions to regulatory proteins

  • has a reversal mechanism for inactivity

• used in replication and gene expression

Histones

protiens made of lysine and argenine that DNA is wrapped around to make nuleosomes

Epigenetics

chemical after-modifications to DNA → usually occur along histone tails (have targets along the chromatin fiber for chemical modifications)

• acetylation: adding acetyl group for gene activation

  • done by: Enzyme: histone acetyltransferase (HAT)

  • removed by: Histone: deacetylase

  • added to lysine (+ charge) - it becomes neutralized

• Methylation: Adds methyl groups to arginine and lysine residues in histones

  • done by: Enzyme methyltransferase

    • DOESNT change charge, just acts as a marker

  • sometimes has positive correlation with gene activity

  • if base C methylated: negatively correlates w/ gene activity

    • forms 5-methyl cytosine

    • can occur at CpG island: Region of DNA where many cytosine guanine dinucleotides present

• phosphorylation: Adds phosphates groups to hydroxyl groups of amino acids serine and histidine

  • done by: enzyme kinase

  • Correlated with chromatin unfolding and condensation

Repetitive DNA

Highly repetitive: Satellite DNA

Middle repetitive:

• tandem repeats

  • microsatellites/STRs (short tandem repeats): repeats dispersed throughout genome

    • Consist of di-, tri-, tetra-, and pentanucleotides

    • Most common in humans is (CA)n with n between 5-50

    • Useful markers for genome analysis

  • VNTRs(variable number tandem repeats)/minisatellites: Repeating sequences 15-100 bps found within/between genes

    • Variation in number results 1-20kbps in size differences

    • Originally used in DNA fingerprinting analysis

  • multiple copy genes/rRNA genes

• interspersed retrotransposons

  • Retrotransposons: Transposable elements generated via RNA intermediate (LINES)

  • SINEs/alu & LINEs/L1: Transposable sequences that are mobile and can relocate within genome

    • Constitute 1/3 of human genome

Centromeres and Kinetochore Proteins

Centromeres

• Separation of homologs during mitosis and meiosis

• Are the primary constrictions along eukaryotic chromosomes

Kinetochore proteins

• Region that binds to spindle fiber microtubules during cell division

Pseudogenes

Large number of single-copy noncoding regions

• DNA sequences representing evolutionary vestiges (parts that no longer have function because of evolution but did before)

• Have undergone significant mutational alterations (insertions and deletions)

• Are not transcribed

**Only a small portion of eukaryotic genome (2–10%) constitutes protein-encoding genes

Transcription

transcribing template DNA into mRNA

• RNA synthesized on DNA template

• Genetic information stored in DNA is transferred to RNA

• mRNA is intermediate between DNA and proteins

Promoter → Initiation → Elongation → Termination → 5' cap → Splicing → Poly-A tail → Mature mRNA

General features of genetic code

• linear form from bases of mRNA

• each “word” is a codon (3 ribonuclotide sequence)

• unambiguous: each triplet specifies only one amino acid

• Degenerate: A given amino acid can be specified by more than one triplet codon

• Contains “start” and “stop” triplets (initiate/terminate translation)

• Comma-less: codons are read with no break

• Nonoverlapping: Any single ribonucleotide within mRNA is part of one triplet

• Colinear: Sequence of codons in a gene is colinear

• Nearly universal: the same “coding dictionary” used by viruses, prokaryotes, archaea, eukaryotes

mRNA, RNA polymerase, and Promoters

messenger RNA

• intermediate for transferring genetic information from DNA to proteins

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RNA pol: Enzyme directs synthesis of RNA using DNA template

• Nucleotides contain ribose, not deoxyribose

• No primer required for initiation

→ In eukaryotes:

• RNA poly I: creates rRNA in nucleolus

• RNA poly 2: makes mRNA & snRNA (and other unique RNAs) in nucleoplasm

  • most responsible for transcription of wide range of genes in eukaryotes

  • Activity dependent on cis-acting elements (explanation) and trans-acting transcription factors

  • RNAP II core-promoter determines where RNAP II binds to DNA

• RNA poly 3: makes 5SrRNA & tRNA in nucleoplasm

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Promoters: Nucleotide sequences as recognition sites for transcription machinery (transcription initiation)

*Interaction of promoters and RNA polymerase regulates efficiency of transcription

Triplet code

• Provides 64 codons to specify 20 amino acids

• Nonoverlapping: Genetic code reads three nucleotides at a time in continuous, linear manner

• Each triplet codon is complementary to anticodon of

  tRNA

Start codon: Methionine (AUG - ONLY codon for met)

• In bacteria: modified form of methionine [N-formylmethionine (fmet)]

• Appears internally in mRNA unformulated

Termination codons: UAG, UAA, UGA

• Do not code for any amino acid/Are not recognized by tRNA

*3rd bp is more flexible (wobble hypothesis)

Frameshift mutations

Result from insertions or deletions of base pair(s)

*causes shift in triplet reading frame

13.3 placeholder

ORF

Open reading frame: “In some viruses, initiation at different AUG positions out of frame with other leads to distinct polypeptides”

→ another start site for other versions of aa chains

Consensus Sequences & Termination/Poly U

DNA sequences homologous in different genes of same organism

→ E. coli promoters have two consensus sequences

• TTGACA and TATAAT (Pribnow box)

  • Positioned at −35 and −10 with respect to the

  • transcription initiation site, respectively

Termination is done by weak pairs of the “Poly U” sequence

• Rho breaks off the base pairs

Transcription in Eukaryotes

• Occurs within nucleus (unlike prokaryotes)

• mRNA must leave nucleus for translation

• Chromatin remodeling makes DNA accessible to RNA Pol

• RNA polymerases rely on “transcription factors (TFs)” to scan/bind DNA

• “Enhancers” and “silencers” and “proximal promoting elements” control transcription regulation (and efficiency)

• Different mechanism of termination

• mRNA processing of pre-mRNAs

Transcription Factors

Transcription factors facilitate RNApol2 binding and initiation of transcription

• General transcription factors: Required for all RNAP II-mediated transcription

• Transcription activators and repressors: Influence efficiency or rate of RNAP II transcription initiation

RNA processing

• transcription → cap → splicing → tail

Splicing

• type of posttranscriptional modification: Introns (non coding regions) removed exons (exon shuffling) joined together (coding regions) in mature mRNA (smaller than initial RNA)

spliceosome: made up of snRNPs

• snRNAs: Small nuclear RNA involved in splicing

• snRBPs: snRNA + proteins; building block of spliceosome

Tail *eukaryotes

• added by poly-a-polymerase: Adds poly-A tail to 3' end.

• poly-a binding protein: Binds poly-A tail; stabilizes mRNA

Translation

Biological polymerization of amino acids into polypeptide chains

requires: Amino acids, Messenger RNA (mRNA), Ribosomes,Transfer RNA (tRNA)

Steps:

• Shine–Dalgarno sequence (AGGAGG) precedes AUG start codon in bacteria

  • “Base-pairs with region on 16S rRNA of 30S small subunit, facilitating initiation”

• Initiation complex

  • Small ribosomal subunit ← initiation factors bind causing → mRNA to attach at codon AUG

  • Combines with large ribosomal subunit

• Elongation

  • mRNA moves into P & A sites (amino acids grow at P site)

  • tRNA exits at E site

• Termination

  • termination sequence enters the A site causing hydrolysis of amino acid chain

  • ribosome subunits dissociate

tRNA

Adapt genetic information (specific triplet codons) in mRNA to corresponding amino acid

• tRNA anticodons complement mRNAs

• tRNAs carry corresponding amino acids

• needs to be charged by enzyme aminoacyl tRNA synthetase

4 attachements sites:

• Amino acid attachment site

• Anticodon

• Recognition site for aminoacyl-tRNA synthetase

• Ribosome recognition/binding site

ribosomes

combination of ribosomal RNA (rRNA) and protiens - 2 subunit: large and small

translation factors placeholders

sl 16

Translation differences in eukaryotes

• Ribosomes are larger and longer lived than bacteria

• Transcription occurs in nucleus

  • 5′ end of mRNA capped with 7-methylguanosine residue

  • Poly-A tail added at 3′ end of mRNA

• Translation occurs in the cytoplasm

• Eukaryotic mRNAs contain A or G three bases ahead of AUG initiator codon, followed by G (Kozak sequence)

  • A/GNNAUGG

• Kozak sequence increases efficiency of translation by interacting with initiator tRNA

One Gene:One Enzyme Hypothesis

• George Beadle showed genes are directly responsible for enzymes synthesis

• Beadle and Tatum showed nutritional mutations in bread mold Neurospora caused loss of enzymatic activity

  • Experiment led to one-gene:one-enzyme hypothesis

*found that most mutants unable to grow on minimal medium could grow on medium with an addition of one supplement on minimal medium. Findings suggested that most mutations affect only a single metabolic pathway/single enzyme.

[check on sl. 25-26]

One Gene: One polypeptide

One-gene:one-protein hypothesis changed to one-gene:one-polypeptide chain hypothesis

Two factors modified the hypothesis:

• Nearly all enzymes are proteins, not all proteins are enzymes

• Proteins have subunit structure with two or more polypeptide chains

Proteins

• Polypeptides: Precursors to proteins

  • Amino acids assembled on and released from ribosomes as polypeptides (Amino acid polymer)

  • held my polypeptide bonds through dehydration

• Proteins: Polypeptides folded up into a functional three-dimensional conformation

• Four levels of protein structure:

  • Primary: Sequence of amino acids

  • Secondary: α-helix and β-pleated sheets

  • Tertiary: Three-dimensional conformation

  • Quaternary: more than one polypeptide chains

    • Protein folding: Co-translational folding by chaperones(other proteins that mediate folding)

    • Diseases of protein folding: Creutzfeldt–Jakob disease, Transmittable brain disorder in mammals, Presence of prions (misfolded proteins) in brain; Others: Huntington’s, Alzheimer’s, Parkinsons.

• Most abundant macromolecules in cells: play diverse roles

  • hemoglobin and myoglobin: transport oxygen for cellular metabolism

  • Collagen and keratin: structural proteins for skin, connective tissue, and hair of organisms

  • Actin and myosin: Contractile proteins found in muscle tissue

  • Tubulin: Basis of microtubule function in mitotic and meiotic spindle fibers spindle fibers

  • Immunoglobulins: Function in immune system of vertebrates

  • Transport proteins: Movement of molecules across membranes

  • Hormones and their receptors: Regulate various chemical activity

  • Histones: Bind to DNA in eukaryotes

  • Transcription factors: Regulate gene expression

  • Enzymes: Most diverse/extensive proteins - specialize in catalyzing (reducing AE) chemical reactions and increase rate of chemical reaction reaching equilibrium

• Amino acids: Carboxyl group, Amino group, R (radical) group bound to central carbon atom

  • R group (side chain) determines type of amino acid: Nonpolar (hydrophobic), Polar (hydrophilic), or positively and negatively charged

Posttranslational modifications

Polypeptide chains modified after synthesis. Modifications crucial to function of final protein product.

Posttranscriptional modification examples:

• N-terminus amino acid removed or modified

• Individual amino acid residues modified

• Carbohydrate side chains are sometimes attached

• Polypeptide chains may be trimmed

• Signal sequences are removed

• Polypeptide chains often complexed with metals

Functional Domains

• Sequences with 50–300 amino acids constitute protein domains

• Sequences constitute protein domains that fold into stable, unique conformations

• Different protein domains impart different functional capabilities

  • Catalytic domains & DNA-binding domains

Mutation, changes, other mutations

Mutation

• An alteration in DNA sequence (Any base-pair change)

  • Transitions: Pyrimidine replaces pyrimidine, or purine replaces purine (pyr: CTU, pur: AG)

  • Transversions: Purine and pyrimidine are interchanged

    • tautomeric shifts: H bond between the wrong pyr/pur

• Single base-pair substitution

• Deletion or insertion of base pairs

• Major alteration in chromosomal structure

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molecular changes:

• Point mutation or base substitution: Change from one base pair to another

• Missense mutation: Results in triplet code for different amino acid

• Nonsense mutation: Results in triplet code for stop codon (translation terminated prematurely)

• Silent mutation: New triplet code still codes for same amino acid

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• Somatic mutations occur in any cell except germ cells (not heritable)

• Germ-line mutations occur in gametes; are inherited

• Autosomal mutations occur within genes located on autosomes

• X- and Y-linked mutations occur within genes on X and Y chromosomes

Classification of mutations by effect on function (not super important- skim)

• Loss-of-function mutations

• Gain-of-function mutations

• Visible (morphological) mutations

• Nutritional (biochemical) mutations

• Behavioral mutations

• Regulatory mutations

• Lethal mutations

• Conditional/temperature-sensitive mutations

Mutation classification + Rates

Spontaneous mutation: natural change in bases

• Arise from normal biological or chemical processes that alter nitrogenous bases (ex: replication, base damage)

• Spontaneous mutation usually very low for all organisms

Induced mutations: from influence of extraneous factors (natural or artificial)

• ex: Radiation, UV light, Natural and synthetic chemicals

Germ Line Rates:

• Average 60 new mutations in offspring

• Number dependent on age of father

Somatic Rates:

• 4-25x higher than germ lines rates

• Somatic mosaicism (early mutation may contribute to a large portion of adult cells having that mutation)

sources of spontaneous mutations

• Mutations from replication: DNA pol occasionally inserts incorrect nucleotides which persist after proofreading

• Errors due to mispairing predominantly lead to point mutations

Replication slippage: If loop occurs in template strand during replication, DNA pol misses looped out nucleotides and small insertions and deletions occur

• more common in repeat sequences (hot spots for DNA mutations)

• Contribute to hereditary diseases (ex: fragile-X, Huntington disease)

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base damage:

Depurination and deamination

• Common causes of spontaneous mutations

• Lead to new base pairing and mutations

Oxidative damage

• Due to by-products of normal cellular processes

  • Superoxides (O2− ), Hydroxyl radicals (OH), Hydrogen peroxide (H2O2)

• Exposure to high-energy radiation

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Transposable genetic elements: DNA elements moving within/between genomes

• Present in all organisms

• Can act as naturally occurring mutagens

• Cause inversions, translocations, double-stranded breaks (creates chromosomal damage)

• Steps: cleaves DNA at ITR region, staggered cuts in target area, inserts transposon inserted by transposase, gaps filled by dna pol + ligase

Sources of Induced mutations

Mutagens: Natural or artificial agents that induce mutations

• Fungal toxins, Cosmic rays, UV light, Industrial pollutants, Medical X-rays, Chemicals in tobacco smoke

Base analogs: look like nitrogen bases - may be incorporated accidently

Alkylating agents: attach alkyl groups, causing issues (mispairing, breaks, etc.)

Intercalating agents: Chemicals w dimensions+shapes that wedge between DNA bps (Example: Ethidium bromide)

• causes base-pair distortions and DNA unwinding

• Usually results in frame-shift mutations

Adduct-forming agents: Mutation-causing chemicals

• Covalently binds to DNA, altering conformation, interfering with replication + repair

  • Acetaldehyde: Component of cigarette smoke

  • Heterocyclic amines (HCAs): Cancer-causing chemicals created from cooking meats

    • 17 HCAs linked to cancers of stomach, colon, and breast

UV light → Pyramidine Dimer

• UV light causes bases of the same strand to bind to each other

Ionizing radiation: causes more X linked recessive alleles

Free radicals: transformation of chemical species with unpaired electrons by radiation

• Alter purines and pyrimidines

• Break phosphodiester bonds

• Produce deletions, translocations, and fragmentation

Trinucleotide repeat sequences

• Short DNA sequences found in mutant genes

• Normal individuals may have a low number of repetitions

• Individuals with over 20 disorders exhibit over 200 repeats

  • Fragile-X syndrome, huntington disease (HD), Myotonic dystrophy (MD)

DNA repair

Repair systems counteract spontaneous & induced DNA damage

DNA repair system: Maintains integrity of genetic material + counteract genetic diseases/cancer

Proofreading and mismatch repair

• DNA pol proofreads - removes, replaces incorrectly inserted nucleotides (3’ to 5’ exonuclease)

• Mismatch repair (if proofreading fails) becomes activated: Mismatches are detected, cut, removed (endonuclease and exonuclease). Correct nucleotide inserted by DNA pol

Strand discrimination - Based on DNA methylation

• Adenine methylase (bacteria enzyme) recognizes DNA sequences and adds methyl group to adenine residues

• Newly synthesized strand of replication remains unmethylated

• Mismatch repair recognizes unmethylated strand and repairs

photoreactivation repair:

base excision repair:

nucleotide excision repair:

• NER pathway defects can cause autosomal recessive diseases

Double-strand break repair (DSB repair): reattaches strands

• Double-strand breaks result in chromosomal rearrangements, cancer, cell death

• Two pathways involved in DSB repair:

  • Homologous recombination repair: uses sister strands and recombination to repair

  • Nonhomologous end joining:

    • Activated in G1 prior to replication

    • Complex of proteins (kinase/BRACA1) is involved in end joining

    • Proteins bind free ends and ligate ends back together

E. coli SOS repair system

Last resort to minimize DNA damage

• DNA synthesis becomes error-prone; inserts random/incorrect nucleotides in places that normally would stall replication

• it induces expression of 20 genes; their products allow replication to move forth

• SOS repair can itself become mutagenic; allows cells to survive with DNA damage (cell would otherwise kill itself)

Ames Test

tests the mutagenic potential of chemical compounds using bacteria

• used bacteria that couldn’t synthesize his (mutegens can restore his)

• tested if his came back on plates w/o histadine in agar

Mobile genetic elements in corn plants found using

Ac–Ds system

• Dissociation (Ds) and Activator (Ac)

  • Two mutations of transposable elements in corn

  • Mobile controlling elements (transposable elements)

• Movement of Ds gene is dependent on Ac gene

• Barbara McClintock

[figure this out]

[figure out what retrotransposons are]

Eukaryote differences from prokaryotes - Transcription Regulation

• Eukaryotic genes on chromosomes w/ distinct location

  • Chromosome territory: In interphase, each chromosome occupies a domain and stays separated from others

  • Interchromosomal domains: Channels between chromosomes that contain little or no DNA

*Chromosome structure is continuously rearranged; transcriptionally active genes are cycled to edges of chromosome territories

• Eukaryotic DNA combined with histones and nonhistone proteins to form chromatin

• Compact chromatin structure inhibits transcription, replication, and DNA repair

Nucleosome modifications

*posttranscriptional modifications:

to histones:

type of gene regulation; involves changes to either nucleosome or DNA

– Histones contain normal histone H2A

– Variant histones (H2A.Z) affect nucleosome mobility and positioning on DNA

– Nucleosome position may repress or activate transcription via gene promoter

DNA methylation:

– Associated with decreased gene expression

– Methylation occurs most often on cytosine of CG doublets in DNA (CpG Islands)

– Methylation can repress transcription by binding to transcription factors of DNA

– Regulated by methylases and demethylases (5’-^mCpG-3’ 3’-GpC^m-5)

cis-acting-sequences

non‑coding DNA element that regulates the transcription of a gene

• promoters (transcription initiation): Nucleotide sequences as recognition sites for transcription machinery

  • Transcription start site, position +1

  • double helix is denatured/unwound to make template strand accessible

    • Located directly next to the regulatory genes (control the expression of other genes)

    • Core promoter: Determines accurate initiation of transcription

    • Proximal-promoter elements: Modulate efficiency of basal levels of transcription

• Enhancers (increase rate of transcription)

  • Located on either side, some distance from, or within gene

  • Important in reaching maximum level of transcription

• Insulators: Found between an enhancer and a promoter for a non-target gene

  • allow some enhancer–promoter interactions and block others

• Silencers (block transcription): Repress the level of transcription initiation

Transcription factors pt 2

*EUKARYOTES

Transcription factors: regulatory proteins that bind to enhancers and promoter elements and fine-tune the level

• Target cis-acting sites of genes regulating expression

• Activators increase transcription initiation

• Repressors decrease transcription initiation

2 functional domains (clusters of amino acids with a specific function):

• DNA-binding domain

  • Binds to specific DNA sequences in the cis-acting regulatory site

• Trans-activating domain

  • Activates or represses transcription by binding to other transcription factors or RNA polymerase

Characteristic domains of DNA-binding proteins

• Helix-turn-helix (HTH): eukaryotic and prokaryotic transcription factors - DNA binding domain

• Zinc-finger: found in many transcription - enables binding

• Basic leucine zipper (bZIP): Allows for protein–protein dimerization

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TFIIA	Stabilizes transcription complex.
TFIIB	Positions RNAP II at promoter.
TFIID	Contains TBP; first factor to bind promoter.
TFIIE	Recruits and regulates TFIIH.
TFIIF	Escorts RNAP II to promoter.
TFIIH	Unwinds DNA and phosphorylates RNAP II.
TBP	TATA-binding protein; binds TATA box.

DABEFH (TFIID → TFIIA → TFIIB → TFIIF → TFIIE → TFIIH)

Formation of RNA Pol II initiation complex

General transcription factors (GTF): Proteins

Required at promoter to initiate basal (find where else basal is used and fix it) or enhanced levels of transcription

Assembly of proteins in specific order forms pre-initiation complex (PIC)

PIC provides platform for RNAP II to recognize transcription start sites

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Transcription activators and repressors change RNA Pol II transcription

• DNA looping delivers activators, repressors, and general transcription factors to promoter

• Recruitment model: enhancers and silencer elements act as donors; affect regulatory proteins at gene promoters

– Chromatin alteration model: looping results in alteration of the chromatin via remodeling complexes

– Nuclear relocation model: looping relocates region to a favorable nuclear environment

GAL gene system

yeast

• Composed of four structural genes and three regulatory genes

• Products of structural genes transport galactose into cell for metabolism

Positive control: Activator protein must be present to turn on gene transcription

are inducible

• Transcription regulated by presence/absence of substrate (galactose)

  • Absence of galactose: GAL structural genes not transcribed

  • Galactose added to medium: Transcription begins immediately

ENCODE

Encyclopedia of DNA Elements Project (ENCODE): Identify all functional DNA sequences and determine how elements regulate expression

findings:

• more than 80 percent of human genome contains regulatory elements (used to be considered junk DNA)

• Enhancer and promoter elements

• Non-coding regulatory RNA

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genome-wide association study (GWAS):

• Identifies genetic variations that are significantly increased in the genomes of patients with diseases

• Many GWASs evaluate single base-pair changes/single nucleotide polymorphisms (SNPs)

  • Do not directly mean disease susceptibility

  • located near the causative issue- the two are inherited together

90%+ of disease-associated variations in the human genome are located within regulatory regions

Alternative Splicing

*EUKARYOTES

different forms of mRNA from identical pre-mRNA (spliceforms)

• expression of one gene = many protiens with similar/diff functions

• increases num. of proteins from one gene (isoforms)

Types of splicing:

• Cassette Exons: skipping an exon region (joining the before and after region)

  • most prevalent type of alt. splicing in animals (40%)

• Alternative splice site: splicing within an exon splice site (shifting where cutting starts)

• Intron retention: including introns - can be used to negatively regulate at post transcriptional level

• Mutually exclusive exons: coregulation where only one exon can be included from a cluster

• Alternative Promoters: having more than one transcription initiation site (diff 5’ ends)

• Alternative polyadenylation: different 3’ poly-A tails created

  • if an exon that signals the poly-A tail is skipped, another exon with the signal is used

An image can be added!!

Regulation of AS

• splicing enhancers/silencers: cis-acting sequences that regulate alternative splicing

• SR protiens: bind to enhancers and activate splicing (recruit spliceosome components)

• hnRNPS (heterogeneous nuclear ribonucleoprotiens): bind splicing silencers = inhibit splicing

• RNA-binding protiens (RBPs): bind to RNA sequences to regulate splicing

*spliceopathies: mutations that affect regulation and cause genetic disorders

mRNA stability

• steady-state level of mRNA: amount of mRNA avaible in cell for translation (determined by transcription and mRNA degredation rates)

• Half life: (t 1/2): half life of mRNA - at what point it degrades

Degredation of mRNA

1) shortened poly-A tail - called deadenylation-dependent decay

2) decapping enzymes removed cap - makes mRNA unstable

3) endonuclease cleaves (removes) mRNA internally

*regulated bu cisacting sequence elements and trans-acting proteins

• adenosine-uridine rich element (ARE): cis-acting sequence that regulates mRNA stability

mRNA surveillance & Nonsense mediated decay

surveillance: degrading mRNAs with premature stop codon that come from a nonsense mutation or RNA pol error

• NMD: most studied surveillance pathway - eliminates mRNAs with premature stop codon

RNAi vs longnoncoding RNAs

RNA interference: sequence specific posttranscriptional regulation - short RNA molecules that regulate gene expression (repress and trigger mRNA degredation)

• studied in lab and developed as pharmaceutical agent

• therapeutic RNAi attacks diseases caused by overexpression

  • potential use in cancer diagnoses + reduces severity of infections by viruses

• in animals:

  • treatment for biral infection, eye diseases, cancer (potential tool in molecular med)

long noncoding RNAs: act as regulators

Regulation of protiens

phosphorylation: most common type of posttranscriptional modification - induces conformation changes → function changes

• kinases catalyze addition of phosphate group to ser, thero, tyro AA side chains

• phosphatases: enzymes that remove phosphates

*ubiquitincan mediates protein degregation