BCHM 3050 exam 1

What's the difference between nuclosides and nucleotides including naming differences?

- Nucleoside \= sugar + base (-sine, -dine)

- Nucleotide \= sugar + base + phosphate (XXP)

Whats the difference between purines and pyrimidines?

- Purine: 5C fused with 6C ring (adenine, guanine)

- Pyrimidine: 6C ring (cytosine, thymine, uracil)

Why is DNA "deoxyribose"?

- The ribose sugar is missing a 2'OH group

What is alkaline hydrolysis?

- RNA is sensitive to mild bases and will catalyze

- Can break its phosphodiester bonds spontaneously

How can UV be used to find concentrations of DNA?

- Nuclotide bases have conjugated double bonds, so they can absorb light

Distinguish between "syn" & "anti" configurations of nucleosides/tides.

- Syn \= base turns on glycosidic bond to face phosphate and 5'OH

- Anti \= base turns to face away from the phosphate and 5'OH

- Pyrimidines hate syn (steric clash between oxygens)

Explain the role of nitrogenous bases in cyclic messaging systems and other metabolic processes

- ATP, GTP, UTP, CTP for energy and metabolism

- Second messengers like cAMP and cGMP that relay messages from primary messengers

- cAMP: metabolite regulation, disease reaction, apoptosis

- cGMP: blood pressure, nerve impulse, stress response in plants, Nitrous Oxide reaction

- Theobromine & Theophylline (Secondary metabolites of cocoa beans/tea leaves, diuretic, cardiac stimulants, vasodilators)

List and describe some adenosine analogs and/or antagonists

- Cordycepin (antibiotic in fungi, prevents polyadenylation)

- Cytokinins (plant hormones for cell cycle)

- Caffeine (antagonistic, can bind to adenosine receptors in brain)

Distinguish between the terms: genome, transcriptome, proteome & and metabolome.

- "-ome" \= body or full collection of something

- Genome \= DNA

- Transcriptome \= RNA

- Proteome \= Proteins

- Metabolome \=Metabolites and macromolecules

Explain what Hershey and Chase did in regards to nucleic acids

- Radioactively labeled proteins (sulfur) and DNA (phosphorus) in T2 phages

- Let phage infect cell, sheared off the coat/virus, then let the phage "reproduce" inside the cell

- Found that ONLY the DNA and its radioactivity entered the cell and were passed down to the progeny, not proteins

- Proved nucleic acids carried hereditary info

What did Chargaff do? Explain his rules

- Studied different species

- Found %A \= %T and %C \= %G

- Showed complementary base pairing, AT, GC

Explain what Rosalind Franklin did and the basic principles of it

- Used X-ray crystallography to prove DNA was in a helix

- When radiation of any kind passes through a regular, repeating structure, diffraction is observed

- Radiation scattered by the repeating elements in the structure shows reinforcement of the scattered waves in certain specific directions and weakening of the waves in other directions

- In certain directions will the scattered waves be in phase and therefore constructively interfere with (reinforce) one another.

- In all other directions, they will be out of phase and destructively interfere with one another

Distinguish between the primary, secondary & tertiary levels of structure in DNA.

- Primary \= nucleotide sequence

- Secondary \= helical shape

- Tertiary \= A, B, Z form (and others)

Describe the structural/molecular/physical characteristics of B-DNA, including shape, size & spatial dimensions, and arrangement of nucleotides & base-pairing (& Chargaff's Rules).

- Double helix shape, RIGHT-handed/clockwise

- Diameter \= 2.4 nm

- Major/minor grooves

- Major \= recognition sites for TIFs for protein interaction

- 10.5 bp/turn

- 1bp \= 0.34 nm of the helix

- A\=T (2 h bonds), C\=G (3 h bonds)

Explain how DNA is metastable

- Phosphodiester bonds are thermodynamically unstable but kinetically stable

- They'll degrade eventually but it takes forever

What are the 4 stabilizing forces of DNA?

- H bonds between base pairs

- Hydrophobic bases

- Van der Waals forces

- Electrostatic interactions

Explain how H bonds between base pairs stabilize DNA?

- Keep the base pairs from slipping

- Have a zippering effect (can form and reform)

- Easier to split AT than GC

Explain how hydrophobic bases stabilize DNA?

- Bases face inward away from the water

- Hydrophilic backbone faces outside toward the water

Explain how Van der Waals forces stabilize DNA?

- Flat/planar bases stack closer together

- Its cumulative effect is more significant

Explain how electrostatic interactions stabilize DNA?

- Backbone has negative phosphates which could destabilize DNA via repulsion

- Avoid this using shielding by Mg2+ ions and positively charged histones

- The charges increase DNA's solubility in water

List some forms of DNA other than B-DNA

- A-DNA

- Z-DNA

- Cruciform

- Triple Helix

- Linear, Circular, Supercoiled

Describe A-DNA and how it's made

- More compact (angle between base and sugar planes is smaller, less perpendicular)

- Still right-handed but wider

- Made when DNA is dehydrated with ETHANOL instead of salt

Describe Z-DNA and how it's made. What is its function?

- Left-handed, forms from excessive CG repeats

- The Gs try to turn syn, but the pyrmidinic Cs don't, the H-bonds hold, so the backbone ends up coiling to make it work

- Functions as replication/transcription regulation

Describe Cruciform DNA and how it's made. What is its function?

- Happen with inverted repeats (palindromes)

- The palindromes form double hairpin loops

- Functions in protein binding to DNA and recognition sites for restriction enzymes

Describe Triple helix DNA and how it's made.

- Happens when supercoiled DNA has an open area

- Hydrophobic bases face water, so the open strand folds back and forms a helix with a closed part of the DNA that was PARALLEL to it, creating a triple helix

- Bases can form more than one set of H-bonds

T/F: All DNA is supercoiled in eukaryotes and prokaryotes at all time

- False: Supercoiled DNA is most prevalent in eukaryotes during metaphase

- It's intermediate in both euks (regular) and proks

List some ways that RNA is different from DNA

- Single stranded (can fold back), has ribose sugar

- Uses Uracil instead of thymine

- Can use modified bases, DNA will mutate with modified bases

- Catalytic (alkaline hydrolysis)

Which is more abundant, RNA or DNA? tRNA or mRNA? rRNA or mRNA?

- RNA more abundant than DNA (more copies and varieties)

- tRNA more abundant than mRNA

- rRNA MOST abundant (60% of RNA)

What are the main types of RNA?

- tRNA (transfer)

- mRNA (messenger)

- rRNA (ribosomal)

- snRNA (small nuclear)

Describe snRNA

- Complexes with protein to make snRNP which function in intron splicing for mRNA processing

Differentiate between eukaryotic and prokaryotic DNA in terms of location, shape, packaging, utilization/amount, length, and reassociation kinetics.

Eukaryotes
-Stored in nucleus
-Uses histones for packaging
-Has a LOT of noncoding regions and repetitive sequences
-Linear, longer
-Slow paced reassociation for unique codes, fast paced reassociation for repeated codes

Prokaryotes
-Not membrane bound, nucleoid
-Uses negative supercoiling for packaging
-Has one chromosome per cell, so it uses majority of its code
-Circular, shorter
-Only one pace for reassociation, slow

Explain the significance of repetitive DNA sequences.

- Can be structural (ex. AT satellite repeats in centromeres)

- Important housekeeping genes are often repetitive for more gene product

- Alu SINES and LINES for transposon jumping

- Intron sequences help genes make different products from one code

Explain the physical organization of eukaryotic genes within the nucleus.

- Chromosomes made of chromatin (DNA complexed with proteins)

- Chromatin held together by nuclear envelope (membrane bilayer with pores for selective import/export of RNA and proteins, but also free diffusion of small molecules)

Differentiate between histones, nucleosomes, chromatin and chromosomes.

Histones
- Positively charged and basic octomers that DNA wraps around

Nucleosomes
- Units of DNA wrapped around histones (~146 bp)

Chromatin
- DNa complexed by histones and other proteins
- Further coiled nucleosomes

Chromosome
- Compacted strand of DNA made of chromatin
- Formed during nuclear division

Distinguish between heterochromatin and euchromatin.

Heterochromatin
- Accessible, relaxed areas in chromatin where proteins can interact with DNA to transcribe/express it

Euchromatin
- Closed or methylated areas in the DNA that aren't accessible to other proteins, not transcribed/expressed

List the different parts of the histone octamer and what H1 does

- Octamer \= H2A, H2B, H3, and H4

- H1 isn't part of the octamer, but its more of a clamp to keep DNA attached to nucleosome and helps bring nucleosomes together to make solenoids

T/F: Histone side-chain modifications affect gene expression by changing DNA base sequence (may or may not be propagated)

- False: they affect gene expression but do NOT change DNA base sequence

Explain how restriction enzymes help map portions of the genome.

- Restriction fragments can be arranged in order to yield physical maps of DNA molecules

- Once a restriction map of a genome is determined, the genome can be fragmented using restriction endonucleases

- These fragments can then be cloned into vectors forming a library for sequence analysis

- Each clone in the library possesses a sequence, and these sequences can be arranged using information from the restriction map

Explain RLFPs and how restriction enzymes come into play

- Restriction Length Fragment Polymorphisms (RLFPs)

- Variations in the length of restriction fragments resulting from action by a specific endonuclease

- PCR and Southern Blotting can use restriction enzymes to map RLFPs to compare DNA amongst individuals

Explain PCR (method, what it needs) and some useful applications.

- Polymerase Chain Reaction

- Exponentially amplify small amounts of DNA in vitro

- Needs: Thermostable DNA polymerase, pair of oligonucleotide primers, dNTPs, and DNA template

- 1. Denature (heat) → Primer annealing (cool) → Extension (heat) and repeat cycle until DNA is amplified enough

- Can be used for southern blotting, gel electrophoresis, DNA libraries, identifying diseases, paternal tests, etc

Generally list the steps of DNA replication and what general proteins/enzymes are involved

- Initiation: Initiation factors, SSB proteins

- Unwinding: Helicase, topoisomerase

- Priming: RNA Polymerase

- Elongation: DNA polymerase, DNA ligase

- Termination

What is meant by the terms semi-conservative, semi-discontinuous, okazaki fragments, origin of replication, bi-directional replication?

- Semi-conservative \= one new strand and one old strand in DNA molecule

- Semi-discontinuous \= one of the new strands (lagging strand) is replicated in fragments, not one fell swoop

- Okazaki Fragments \= lagging strand fragments of replicated DNA

- Origin of Replication \= where DNA first splits and replication begins

- Bi-directional Replication \= 2 forks, replication going opposite ways of the origin simultaneously

Describe prokaryotic DNA polymerases, including their subunits?

DNA Pol III: main replicative enzyme, holoenzyme of many subunits
- Alpha \= 5' to 3' elongation
- Epsilon \= 3' to 5' exonuclease repair
- Theta \= unknown
- Beta \= sliding clamp for processivity
- Delta, Delta', Chi, Gamma \= clamp loading machinery

DNA Pol I: fills in gaps, repairs mismatches, replaces RNA primer

Describe eukaryotic DNA polymerases, including their functions? What is PCNA? What is FEN1?

- DNA Pol Epsilon \= main replicative enzyme (leading strand)

- DNA Pol Delta \= replicates lagging strand, replaces primers

- DNA Pol Alpha \= RNA Primase (initiation)

- PCNA \= complex of polymerases, not a real sliding clamp, adds processivity

- FEN1 \= removes RNA primer

Identify the functions of the other enzymes involved in replication(i.e. topoisomerase, ligase, helicase, primase etc.)

- Topoisomerase \= Nicks and relaxes the DNA ahead of the fork to relieve tension and prevent supercoiling

- Ligase \= Seals Okazaki fragments and other gaps in the DNA

- Helicase \= Unwinds the DNA

- Primase \= Lays down an RNA primer so DNA polymerase can elongate from there

- SSB \= Bind to the open DNA to prevent re-annealing and protect DNA from nuclease degradation

Describe the DNA polymerase reaction as it pertains to base incorporation.

- DNA polymerase "feels" out for the right base

- Can tell when the base shape is wrong or doesn't fit

- Sometimes makes mistakes, but proofreading will usually correct it

What is a replisome?

- Whole replication machinery

- DNA polymerases + Primosome (Primase + helicase) + SSB

Describe the bacterial replisome

- SSB proteins, Initiation factors (DnaA)

- Helicase (DnaB), topoisomerase

- RNA Primase (DnaG)

- DNA Pol I & III

- DNA Ligase

- Ter sequences/binding proteins

Describe the mammalian replisome

- SSB proteins

- Helicase (MCM), topoisomerase

- RNA Primase (Pol Alpha)

- DNA Pol Epsilon/Delta

- FEN1, PCNA

- DNA Ligase

- Telomerase/Telomeres

When in the cell cycle does replication occur for eukaryotes? What happens prior?

- S- phase

- Before S-phase, during M, G1, and G2, the origin of replication is marked, then licensing factors bind to it (MCM, CDK)

- These factors have to fall off right before replication begins

- If they stay on after replication, then the gene could be replicated again which would cause genetic issues (extra copies of chromosomes/genes

Explain the significance of telomeres in replication.

- Telomeres cap the ends of eukaryotic chromosomes, vital to their survival

- With each replication, the 5' end is hard to complete and often has a chunk left off

- Without telomerase enzyme, the telomeres would continue to rapidly shorten until the cells dies

- Telomerase adds repetitive TTAGGG to the ends to fill in the gaps and keep telomere length constant as long as it can

Describe chromatin rearrangement/remodeling as it pertains to DNA replication.

- Chromatin structures (histones, SSB) have to be removed before the fork gets to the DNA there

- Have to be added back to the DNA behind the fork to reform the nucleosome

Explain the difference between bacterial/mammalian cell replication in comparison to viral replication (retroviruses)

- Virus takes tRNA and uses it as a primer, then extends from it

- Uses Rnase H to cut extra viral RNA away in the genome and circularize

- Reverse Transcriptase (RNA dependent DNA polymerase) replicates the viral RNA into DNA (rolling circle method)

- RNA strand is kicked off, and DNA doubles up to make two strands

- Incorporates into the host genome via Integrase enzyme to begin infection

Define "mutation" and exemplify or describe the range and types of mutations that can occur, and the mechanisms of how they occur including Loss of function, Gain of function, Dominant negative, and lethal, gene aberrations, and base deletions/insertions/duplications

- Mutation \= Permanent change in the structure of DNA or chromosomes

- Loss of Function \= Gene has less or no function (null)
- Amorphic, usually recessive or result of haploinsufficiency

- Gain of Function \= Gene has more or a new abnormal function
- Neomorphic, often dominant

- Dominant Negative \= Gene acts against its normal function
- Antimorphic, often dominant or semi-dominant

- Lethal \= Phenotype is incapable of reproduction or sustaining life

- Gene Aberrations \= Fragment insertions/deletions, Inversions, Duplications, Ploidy changes
- Often from crossing over issues

- Base deletions, insertions, or substitutions
- Often from DNA replication error (spontaneous or induced chemical changes)
- Silent, Missense, and Nonsense

What are the 3 main origins of mutation?

- Replication errors

- Chromosome alignment/separation errors

- Spontaneous or induced chemical changes in base structure

Explain tautomeric shifts and what kind of mutations they cause after rounds of replication

- Spontaneous isomeraization of base (keto --\> enol or amino --\> imino)

- Cause transition mutations (like replaces like) after 2nd round of replication

What kind of mutation is sickle cell? Explain

- Missense

- E6V mutation: GAG for Glu (E) is changed to GUG for Val (V)

- Changes AA from polar to non-polar, changes shape of hemoglobin protein

Describe the 6 main types of mutagens (damaging agents), how they work, and how they might be repaired.

- Ionizing radiation (X and gamma ray)

- UV Radiation
- Two adjacent thymines form a photoproduct (cyclobutane thymine dimer or 6-4 linkage)
- Use photoreactivation or NER to fix it

- Methylating agents (MNNG)
- Alkyl group is added onto a base
- Usually O6-meth-guanine
- Use alkyltransferases to fix it

- Cross-linking reagents (cisplatin)

- Oxidative reagents
- ROS (reactive oxygen species) causes oxidative product to form
- Use BER to fix

- Bulky adducts (hydrocarbons)
- Fix with NER

What are some endogenous reactions that cause mutations?

- Depurination

- Deamination

- Base oxidation

- Base alkylation

List 7 types of DNA repair mechanisms

- Photoreactivation (direct)

- Alkyltransferases (direct)

- NER (nucleotide excision repair)

- BER (base excision repair)

- Prokaryotic mismatch repair

- Double Strand Break repair

- Daughter Strand Gap repair

Explain how photoreactivation and alkyltransferases work

- Photoreactivation (direct)
- Photolyases use FADH and MTHF to break pyrimidine bonds
- Polymerase and ligase fill in the gaps

Alkyltransferases (direct)
- Alkyltransferases take the alkyl group and put it on themselves, then signal for more alkyltransferases to be made
- They can only be used once, then they degrade

Explain how NER works

- Repairs thymine dimers and bulky adducts

- Bend DNA, use endonuclease to cleave and cut out dimer/adduct on a single strand

- Fill in the gaps with helicase, polymerase, and ligase

- Euks cut out ~30 nucleotides while proks cut out ~11

Explain how BER works

- Used to remove uracil mismatching or oxidative damage

- UNG pulls out dUTP residues by cleaving glycosidic bond, leaving behind an AP site

- Polymerase and ligase fill in the gap

- OGG1 enzyme does the same for when 8-oxoguanine causes a transversion mutation
CG → C-oxoG → A-oxoG → AT

Explain how mismatch repair in prokaryotes works

- Parent strand is methylated while daughter isn't (right after synthesis)

- So they can tell which strand is correct and which one has the mutation

- MutS scans and binds to it, MutL and MutH come in

- Use ATP hydrolysis to bend DNA and cleave it out (follow with polymerase and ligase to fill in gaps)

Explain how DSB repair works

- DSB lesions caused by ionizing radiation or replication stalling, can occur during crossing over

- Most lethal form of DNA damage (destroys physical integrity of chromosome)

- HR (homologous recombination) can only happen during S or G2 when a sister chromatid is available

- NHEJ (non-homologous end joining) is more efficient if the ends can be rejoined EXACTLY at the site of damage

- MRN trims at 5' ends of each strand, RPA/ssb bind to the DNA, then replaced by rad51 which holds it while looking for homologous sequences on sister chromatid to start synthesis

- BRAC1&2 are associated with it

Explain how daughter strand gap repair works

- Replicated part of an incompletely replicated chromosome is used as a repair template

- Process depends on RecA (rad51 homolog)

- Doesn't fully repair damage but allows replication to continue and lets the error get fixed afterward

Explain site specific recombination and what it does

- Allows lysogeny in lambda bacteriophage

- Sticky ends let it circularize then re-linearlize after entering host genome

Explain the Holliday model of recombination

- DNA is nicked at the same site on two paired chromosomes

- Next partial unwinding of the DNA allows strand invasion

- Enzymatic ligation generates a crossed-strand intermediate (Holliday junction)

- The cross-strand structure can move along the duplex

- The Holliday junction isomerizes

- Strand breakage generates a recombinant (all 4 strands) or nonrecombinant heteroduplex

- Relies on RecA to search for complementary strand

- Prokaryotes rely on RuvABC for branch migration and movement along the junction

- RecA uses ATP to promote the pairing of homologous DNA sequences

- RecA recombination sites are recognized by the RecBCD complex

Describe the processes involved in gene rearrangements. What are some of the products of these rearrangements? (VJ joining, transposon)

- Splicing (VJ joining) leads to antibody diversity depending on which immunoglobulin needs to be made

- Transposons use transposase to jump genes and can cause insertions or deletions

What are gene amplifications? Explain how they're related to drug resistance unequal crossing over

- When a duplication or rearrangement causes a gene to be overexpressed

- Two modes of gene amplification can lead to drug resistance
- Cells can become resistant to methotrexate (inhibitor of dihydrofolate reducatase (DHFR))
- Tandem duplication DHFR gene generates a chromosome with multiple copies of the gene in a homogeneously-staining region (HSR)
- A DNA segment may be excised to form minichromosomes called double-minute chromosomes
- In both cases, resistance is conferred by overproduction of the DHFR enzyme

- Gene amplification may occur via unequal sister-chromatid exchange (unequal crossover)

Distinguish between the template strand and the non-template or coding strand, and the direction of template reading vs. transcript synthesis

- Template strand \= non-coding strand, what RNA is synthesized from

- Coding strand \= non-template strand, exactly the same as the mRNA transcript except it has Ts instead of Us

- Polymerase reads the template strand in the 3' to 5' direction so it can synthesize the mRNA in the 5' to 3' direction

Define "promoter" in relation to its structure, function and location in DNA, including the role of consensus sequences, and base sequence characteristics.

- Promoter \= AT rich consensus sequence at beginning of gene, recognized by polymerase as the start site for transcription

- Varying levels of strength (how well polymerase can recognize it)

- Different promoters can specify which tissues express certain genes and at which developmental stage a gene will be expressed

Describe the role of promoters in prokaryotes

\- -35 → -10 → +1 (start) region

- Transient sigma factor recognizes the promoter, gets transcription machinery situated on it while alpha subunit of polymerase binds to it

- Sigma has to let go for elongation to start

Describe promoters in eukaryotes (TATA, GC, CAAT)

- TATA box \= start site, similar to prokaryotic -10 region, hs to be there but other regulatory sites can be upstream of it

- GC box \= indicates its a housekeeping gene, needs to be constantly transcribed

- CAAT box \= gene is a strong promoter

Describe prokaryotic transcription initiation, including the parts of RNA polymerase

- RNA pol is a holoenzyme of 5 polypeptides

1. Beta' \= binds to DNA

2. Beta \= catalytic site

3. Alpha \= binds to promoter, assembles/regulates

4. Omega \= structural role

5. Sigma factor \= recognizes promoter, allows polymerase to situate at right site

- Once polymerase transcribes ~10 NTP, sigma falls away and elongation begins

What are lampbrush chromosomes?

- Once enough DNA is freed, multiple RNA polymerases can bind to the DNA and begin transcribing, making "bristles" of transcripts to form "Lampbrush Chromosomes" (not common in eukaryotes)

Describe Rho factor dependent transcription termination

- Right when transcript is made, rho follows RNA pol down the DNA waiting for it to pause at the termination sequence

- Then it knocks the bonds between the polymerase's active site and snatches the transcript away (NO stem loop)

Describe Rho factor independent transcription termination

- Happens without rho factor

- RNA polymerase reaches terminator regions of DNA

- Terminator: Poly A (consensus\=AAUAAA) regions of (RNA) DNA that code for "hairpin" mRNA structures.

- Hairpins "contract" length of message, complementarity is lost, transcription complex is destabilized

- mRNA & polymerase fall away

Describe eukaryotic RNA polymerases

- 3 RNA polymerases, NONE can initiate transcription and require TFs (transcription factors)

- RNA Pol I \= in nucleolus, transcribes large rRNAs

- \***RNA Pol II \= in nucleus, transcribes mRNA and snRNA

- RNA Pol III \= in nucleus, transcribes tRNAs and small 5s rRNAs

Describe eukaryotic transcription initiation

- TFs bind to TATA box, then signal for pol II to bind to the promoter, TFIIH activates polymerase via phosphorylation to start transcription

- TBP binds to TATA box, bends it 90 degrees to open it up

- TAFs (TBP associated factors) combine with TBP to make TFIID

- TFIIB has b-linker element to open the DNA

- TFIIA stabilizes TBP/DNA

- Enhancer regions and proteins bound at promoter communicate via multiprotein complex called mediator

Describe eukaryotic transcription termination

- RNA pol II transcribes past the end of a gene through one or more TTATTT signals and then falls off

- The pre-mRNA with the AAUAAA signal is cleaved 11-30 NTPS downstreams of these sites

- Polyadenylation at 3' end signals END of transcription

Describe polyadenylation and its purpose

- 100-250 adenines are added to the 3' end of the transcript by a different RNA polymerase while Pol II is stalling at the termination sequence

- Poly A tail stabilizes transcript so it can leave nucleus without being degraded

Describe guanine capping

- Methylguanosine is added to the 5' end of the transcript

- First few bases at the 5' end have their 2'OH groups methylated which signals for the cap

- Cap saves transcript from being degraded by nucleases

- **Some viruses can make transcripts in eukaryotic hosts and get through the nucleus sans 5' cap just fine

Define and/or relate the terms mRNA splicing, introns, exons including the functions of each and the mechanism by splicing is achieved.

- Small nuclear ribonucleoproteins (snRNPs) all start with U, help to form the loop/lariat in pre-mRNA

- 2'OH of the branchpoint nucleophilically attacks the phosphate at the 5' end of the splice site to form a lariat, then releases the 5' exon

- 3'OH of the 5' exon attacks the phosphate of the first 3' exon nucleotide to form the spliced mRNA

- Intronic sequence gets snipped out and degraded

- Pre-mRNAs have short sequence at ends of introns as splicing signals

- Spliceosome \= snRNP + pre-mRNA complex

Distinguish between constitutive vs. inducible genes. Explain constitutive and non-inducible mutations

- Constitutive \= Constantly being transcribed, always making product

- Constitutive phenotype: Mutated when its being made at high levels despite inducer not being there (recessive)

- Inducible \= Genes being made at very low levels

- Non-Inducible Phenotype: Mutated when they're still at low levels despite inducer molecule being present (dominant)

Explain positive vs negative regulation in operons

- Positive \= Regulatory molecule is an ACTIVATOR
- Activation \= activator is active/bound, transcription is ON
- De-activation \= activator is inactive/not bound, transcription is OFF

- Negative \= Regulatory molecule is a REPRESSOR
- Repression \= repressor is active/bound, transcription is OFF
- De-repression \= repressor is inactive/not bound, transcription is ON

Define & exemplify the term "operon"

- Single transcriptional unit

- Series of genes + promoter + operator

Describe the components of the lac operon and what happens when the operator/repressor genes are mutated

- lacZ \= B-galactosidase

- lacY \= B-galactosidase permease

- lacA \= thiogalactosidase transacetylase

- lacI \= repressor molecule that regulates it (negative)

- lacO \= the operator

- Mutations at operator or repressor/activator cause the whole operon to malfunction, but a mutation in one of the structural genes will only affect the molecule it makes

What are the 3 inducers of the lac operon? Which one is important for in vitro study of the lac operon?

- Lactose, allolactose, and IPTG

- IPTG: beta-galactosidase can't cleave the glycosidic bond in it, so its concentrations stay the same, allowing us to study the lac operon in vitro without disturbing the genes

Describe repression in the lac operon and when it would occur

- Repressor binds to operator, keeps RNA polymerase from binding, TRANSCRIPTION OFF

- Only happens if bacteria doesn't need the lac operon to be on (have a better source of food)

- OR if its a non-inducible mutation (repressor binds even when inducer is present)

Explain how de-repression in the lac operon works?

- Inducer binding to site on repressor lowers the repressor's affinity for DNA, INACTIVATING the repressor

- Allows RNA polymerase to bind to operator, TRANSCRIPTION ON

Explain how positive regulation (catabolite repression) in the lac operon works?

- Prokaryotes prefer glucose over lactose

- CRP \= cAMP Recruiting Protein, promotes lac gene transcription by exposing promoter to RNA polymerase, helps recruit RNA polymerase

- Binding cAMP to CRP increases affinity for DNA

- High glucose \= low cAMP → Inactive CRP → lac operon not fully transcribed

- Low glucose \= high cAMP → Active CRP → lac operon fully transcribed

List modes of gene regulation in Eukaryotes

- Genomic control

- RNA processing

- Regulation of RNA out of Nucleus

- Translational Control (message stability)

- Signal Transduction

Explain the genomic control aspect of eukaryotic gene regulation

- Methylation, acetylation, translation factor (TF) availability, chromating remodeling

- TFs are the limiting factor in transcription, NOT RNA polymerase

- Generally acetylating histones promotes transcription via euchromatin (expanded)

- Generally methylating histones deters transcription via heterochromatin (condensed)

Explain how methylation of lysine 9 affects chromatin

- When lysine 9 is methylated, it brings in HP1, PRC recruits deacetylates to remove acetyl groups and heighten ionic interactions between DNA and histones

- Anything that neutralizes the charge on the lysine residue of the histones will make the histones less positive and LESS likely to bind to DNA, which causes euchromatin to form

- So HEIGHTENING ionic interactions will keep the histones and DNA together to make heterochromatin

T/F: Methylation always \= heterochromatin and acetylation always \= euchromatin

- False

- Not all methylation \= heterochromatin

- Ex. H3K4 methylation is a transcriptional activating signal which makes euchromatin

Chromatin Remodeling complexes modify histones and move them out of the way via \_______________.

- ATP hydrolysis

Describe bromodomains and chromodomains

- Bromodomain \= interact with acetylated lysine residues

- Chromodomain\= interact with methylated histones

List and describe the 4 Families in ATPase Domain

- SWI/SNF \= Has a bromodomain, moves histones out of the way

- INO80/SWR1 \= Replaces histone 2A with 2AZ (also involved in DNA repair at stalled forks)

- CHD \= Has a chromodomain, important at H3K4

- ISWI \= Helps form heterochromatin (transcription repression)

Explain the relationship between epigenetics and methylation. How are they related to diseases? Give some examples

- Histone modifications and phosphorylation of the C-Terminal Domain (CTD) of the RNA pol II

- Histone modifications are also altered as a consequence of CTD phosphorylation changes

-\****We inherit histone modification patterns

- Coffin-Lowry Syndrome
- Gain of function mutation in RSK2 gene (histone phosphorylation)
- X-linked dominant, affects males more

- Rubinstein-Taybi Syndrome
- Mutation in CREB-binding protein (histone acetylation)