BIOMG 3320 Final

Example of Roles for microRNAs

- works with fluorescent neurons - mutation: left neuron converted to right neuron - done through deletion of a microRNA - microRNAs are additional gene regulation

PCR

1. strand separation through heat 2. add primers and cooling down 3. add thermostable DNA polymerase, Mg2+, and dNTPs

Translation

1. string of bases on DNA creates identical mRNA base sequence but with U instead of T 2. ribosomes perform translation and synthesize protein 3. tRNA comes in bound to amino acid into ribosome with base pairing between tRNA amino acid and bases

LINC RNA Mechanisms

1. transcriptional activator 2. transcriptional repressor 3. transcriptional guide 4. scaffold for chromatin modification complex (most common, allows complex to come together) 5. RNA degradation regulator

Cell Differentiation

- totipotent: stem cells can be anything - unipotent: cell can only be itself

Code

- 16 possibilities of base pairs, so need more than 2 bases for an amino acid - codon: 3 letter base sequence that codes amino acid - code is colinear: nucleotide order corresponds to amino acid order - non-overlapping: insertion and deletion mutations changes non-overlapping code greatly but overlapping not so much and a combination of the mutations restores the reading frame

History of Genome Sequencing

- 1970s: phages sequenced (~5kb) - 1995: first bacterial genome (~0.5mb) - 2001: first human genome (3200mb) - present >> 1,000 sequenced bacterial genomes, hundreds of eukaryotic mammals, cost of sequencing is must cheaper and easier

One Replication Cycle of E. coli Involves

- 2 replication forks - 2 leading strands - 2 lagging strands

Base Hydrolysis of RNA

- 2' OH can undergo spontaneous hydrolysis (RNA getting spontaneously broken down with water) so add lots of base to interact with water - if undergoes spontaneous hydrolysis then becomes unprotonated and the O can attack the phosphodiester bond - this breaks RNA so it's more fragile

DNA Sequencing: Sanger Sequencing

- 2' deoxy ATP creates 2'-3' dideoxy ATP (w/o OH) which are chain terminating nucleotides so once dideoxy is incorporated, cannot extend anymore since there is no 3' OH - DNA template, primer radiolabeled all 4 bases with one base that has a dideoxy version at low concentration - rxn proceeded and product ran on a gel - radioactive bands must have chain terminating nucleotide which gives you all that base's locations - repeat for all the bases and read off the sequence

Splicing: 1st Transesterification

- 2'OH on branch A performs nucleophilic attack on 5' most nucleotide - creates a new phosphidester bond - A becomes branched

How many incorrectly added amino acids in 20 nucleosomes?

- 20 nucelosomes with 8 histones: 160 histone proteins - 1 histone = 20kDa and 1 amino acid = 0.1kDa so 1 histone = 200 amino acids - 200 amino acids * 160 histones = 32,000 amino acids - error rate is 0.1% so 0.001*32,000 = 32 amino acids

Paused Polymerase and Promoter Escape

- 20-60 nucleotides in, Pol II pauses after synthesizing - Pol II and DSIF and NELF (negative elongation factor) causes Pol II to pause - PTEFb (positive elongation factor) releases NELF by adding phosphates to NELF and DSIF (inhibits) allowing Pol II to continue

(Not) Paused/(un)Expressed Genes

- 25% of all genes: not paused, unexpressed - 45% of all genes: not paused, expressed (not regulated) - <1% of all genes: paused, unexpressed (heat shock genes, can become default in right conditions) - 30% of all genes: paused, expressed (pause release is RDS, regulated)

Splicing: 2nd Transesterification

- 3'OH on first nucleotide performs nucleophilic attack on 3' most nucleotide - creates linkage between exons

Regulation of Chromatin

- 30nm to 10nm is dynamic (convert to 10nm to transcribe) - interaction of DNA with histone is dynamic - must regulate: conversion from 30nm to 10nm and exposure of naked DNA

Regulation of Translation Initiation in Bacteria

- 30s binding at ribosome binding site is a crucial step - most common regulation: if low nutrients, protein (competes with small subunit) binds close to start site and prevents association of 30s and ribosome binding site

Human Genome Breakdown

- 3E9 base pairs, 23 chromosomes, 25,000 genes - 45% transposons: 20% LINEs (long interspersed nuclear elements, especially retrotransposon which is able to copy itself and place the copy somewhere and has 850,000 copies but many are not functional), 13% SINEs (short interspersed nuclear elements, 1,500,000 copies and rely on other transposons for amplification and accumulation), 8% retrovirus derived (450,000 copies that are nonfunctional) - 25% misc: 3% SSR (simple sequence repeats), 5%: SD (segemental duplicatons), 17% unknown - 30% genes of which 28.5% are introns and non-coding and 1.5% are exons

Translation Initiation in Eukaryotes and Cancer

- 4E-BP gets phosphorylated by kinase and when it's phosphorylated it is unable to inhibit - with the phosphorylation cycle, the phosphorous group can be removed and then 4E-BP can inhibit translation - kinase that phosphorylates is mTOR and it is a target of anticancer drugs which inhibit it

Regulation of Translation Initiation in Eukaryotes

- 4E-BP is an inhibitor that binds to mRNA close to 5' cap and ElF4E - once 4E-BP is engaged, prevents associations of other initiation factors so the ribosome is unable to engage - this is highly regulated: senses availability coupled to inhibitor

Polymerase Channels

- 5 channels: 1. RNA exit channel 2. DNA enter channel 3. template DNA exit channel 4. non template DNA exit channel 5. nucleotide enter channel

Transcription Factors in Eukaryotes

- 5% of proteins - interact with specific enhancers, govern certain genes - in family: bind same way but different members bind slightly differently - ex: leucine zipper that binds major groove has sequence specificity that derives from amino acid from family members

Topoisomerase Form Covalent DNA-Protein Links

- 5'-phospho-tyrosyl DNA protein linkage - Tyr residue in active site attacks phosphodiester bond in backbone to create cleavage

LINC RNAs in Detail

- >1000 identified - >200 NT long - transcribed by RNA Pol II, spliced, polyadenylated, capped - functions unknown - employ diversity of mechanisms

Discovery of First DNA Polymerase

- Arthur Kionberg in 1957 - radioactively labeled nucleotides and combined with DNA template and E. coli extract which resulted in radiolabeled DNA and excess nucleotides - determined if the rxn work by using TCA precipitation to precipitate DNA out and this would cause a difference in the radiation levels - then fractionized the E. coli sample to determine the enzyme

lac operon

- CAP site = activator - operator - CAP + operator = promoter - lac Z (galactosidaze) and lac Y (permease): uses lactose as food source - low lac operon growth on glucose: glucose and lactose available, basal level of lac operon expression - repress lac operon growth on glucose: glucose available lactose is not, no transcription - express lac operon growth on lactose: no glucose but lactose is available, activated level of transcription

Transcription in Bacteria

- DNA non template strand (same sequence as RNA) -> DNA template strand (3'-5' base pairing with RNA) -> RNA transcript (5'-3') - lots of genome is for transcription regulation

E. Coli has Several DNA Polymerases

- DNA Polymerase 1 is not the replicative polymerase because it has low polymerization rate (20 nucleotides per second but should be 2,000 nucleotides per second), low processivitiy (3-200 nucleotides before dissociation) and without it the cell is still able to function - in E. coli: DNA Pol II, Pol III, Pol IV, Pl V and each is specialized for different roles in the cell

DNA-Histone Interface (Compaction, Hbonds, Groove)

- DNA binds in left handed spiral that circumnavigates the histone complex 1.65 times - bound to nucleosomes results in a 7 fold compaction - 40 H bonds between histones and DNA (positive histone with negative oxygens in minor groove) which facilitates DNA binding to histone core - binds in minor groove so sequence specificity is small and nucleotides can be formed in most chromosomal regions

Chromatin Again: Histone Core and Chromatin Functions

- DNA bound/packaged by histone proteins - nucleosome = DNA wrapped around a histone - linkers: DNA connecting nucleosomes - histone core has 4 proteins, 2 of each making it an octomer - functions of chromatin: 1. packaging DNA 2. contribute to gene regulation (histone code) since tail regions stick out of nucleosomes and receive covalent modification - euchromatin: unpacked, transcription heavy - heterochromatin: packed, transcription light

DNA Packaged Into Chromosomes

- DNA fits 50mm into 5um - highly compacted (compaction ratio about 10,000) - first wrapped around histone to make nucleosome which helps with DNA packing - then nucleosomes self assemble into 30nm fiber - next 30nm fiber wraps into larger loops

Why do drugs that deplete dNTPs but do not alter level of NTPs promote single stranded DNA in cells with moving replication forks?

- DNA polymerases would stall because not enough dNTPs - helicase would continue to separate strands because ATP is still available - this uncoupling of DNA and helicase results in single stranded DNA

Homologous Recombination Roles

- DNA repair to prevent death by double stranded breaks - genetic diversity in meiosis after many random breaks are created

DNA Polymerase

- DNA synthesis: assembling nucleotides into DNA molecule using a polymerization rxn and must be synthesized by nucleotide polymerizing enzyme

Methyl Transferases

- DNMT: DNA Methyltransferase - de novo methyltransferases: establish methylation patterns in early development - maintenance methyltransferases: methylation patterns retained during cell division by recognizing hemi-methylated state and add missing methyl group (ie. recognize C across from G that is unmethylated but next to methylated C) - when methylation occurs, more factors are recruited such as deacetylation and chromatin remodeling which further repress the gene

Initiation of DNA Replication in E. coli

- E. coli initial strand separated at a single origin of replication (OriC) - initiator protein DnaA binds repeated 9-mer elements in origin of replication and distort/unwind adjacent region of DNA with 13-mer repeats (13-mer is to the left of 9-mer)

araBAD operon

- E. coli metabolize arabinose - additional binding sites - no arabinose: AraC binds regions and promoter not accessible - arabinose available: change promoter conformation by binding to AraC and polymerase can activate and can also have CAP binding site with cAMP if no glucose and two activators at same promoter

Comparing Gene Structures

- E. coli: densely packed, lots of genes - yeast: densely packed - drosophila: some splicing, fewer genes - humans: only two genes in this region, lots of splicing and transposons

Bacterial and Eukaryotic Cells

- bacteria: much smaller, one circular DNA molecule with 5 million base pairs and 5,000 genes - eukaryotic: 10 times bigger, has a nucleus where the genome is found, multiple linear DNA molecules (chromosones), 10 million-1 trillion base pairs. 6,000-30,000 genes

Chromatin and Transcription (FACT complex)

- FACT complex: facilitates chromatin transcription by quickly unwrapping and then rewrapping nucleosomes 1. disassembles nucleosomes 2. assembles nucleosomes

Hayflick Limit

- Hayflick limit: intrinsic countdown clock that limits # of cell divisions and this clock could be a telomere - telomere in younger people is longer than in older people - cells that can't be cultivated for many generations have limited telomerase activity - cells with proliferative capacity have increased telomerase activity

Initiation Factors for Bacteria Translation

- IF3: prevents reassociation (leaves to allow association) - IF1: prevents tRNAs from binding in A site - IF2: facilitates binding of fMet with small subunit

Long Intergenic Noncoding (RNAs, LINC)

- LINC RNA is transcribed (between genes) but function unknown - histone marks identify LINC RNAs

Codons and Amino Acids

- Nirenbirg: cell-free translation system (cell extract + ATP + one amino acid) - start with poly(U) + phenylalanine which creates a polypeptide but with other amino acids no polypeptide is created - can determine what UUU, AAA, CCC code (GGG codes nothing) - next, vary ratio of two bases for random polymer (ie. lots more A than C) and then measure incorporation of amino acids - Asn, Gln, Thr are more frequent so must be A2C compared to His which is less frequent and must be AC2 (also already know AAA and CCC) - Khorana: create synthetic RNA with defined sequences (ie. ACACACAC results only in Thr and His) but using Nirenbirg also know that CAC must be His and therefore Thr must be ACA - Nirenbirg then used purified ribosomes, synthetic RNA, and aminoacylated tRNA (binds specifically) to see what bound most stably and solved 61 codons (other 3 codons are stop codons)

Mischarging of Amino Acids into tRNAs major source of errors in translation?

- No! - error rate of translation: 0.1-0.01% - rate of mischarging: <0.005%

DNA Pol III Major Replicative Polymerase: Subunits

- Polymerase III molecular machine is optimized to replicate the E. coli genome - it is a holoenzyme which achieves replication of leading and lagging simultaneously - polC is the subunit with polymerization activity - processivity of polC alone is quite low (100 base pairs) so to achieve high processivitiy there is a sliding clamp (processivity factor, protein complex) that prevents dissociation

Release Factors in Termination

- RF1 & RF2 activate hydrolysis of polypeptide from peptidyl-tRNA - RF3 stimulates dissociation of RF1 & RF2 from ribosome - release factors mimic tRNA

Retroviruses and Reverse Transcription

- RNA as template to make DNA: EXCEPTION TO CENTRAL DOGMA

RNA as Genetic Material

- RNA not stable in aqeous solution but similar potential for information - NO ORGANISM uses RNA as genetic material, but viruses use RNA as genetic material since they are very short (longer genome means hydrolysis is a bigger problem)

RNA World Hypothesis

- RNA only biomolecule that functions as genetic material and as an enzyme - evolution: RNA picked out that could replicate itself, fold into unique stable structure - technically unprovable hypothesis

Chemistry of RNA

- RNA very similar to DNA with some exceptions: 1. has OH at 2' instead of H 2. RNA uses uracil instead of thymine (replace methyl with H) 3. mostly single strand - many types of mRNA and tRNA

TFIIB, TBP, DNA

- TBP binds DNA in minor groove and distorts DNA - binding at the TATA box induces a 90º turn of DNA - this unusual shape defines the promoter - BRE: TFIIB binds to BRE (B recognition element), close to TBP

RNA Pol II Promoters and Initiation

- TFIID binds DCE and it contains TBP 1. TFIID on promoter first (a strong promoter contacts with TFIID effectively) 2. TFIIA recruited 3. TFIIB recruited 4. Pol II recruited to promoter due to TFIIB and TFIIF interaction 5. TFIIE and TFIIH recruited: helicase activity that uses ATP to unwind DNA or remodel protein interactions, also used to remodel to open complex and set up signals to control downstream through addition of phosphates onto Pol II tail (change pattern of phosphates changes Pol II behavior)

DNA Repair

- essential for life and disease prevention - large portion of genes in genome code for proteins in repair pathways

Mechanics of lac operon

- activated level through allosteric regulation to change conformation: low glucose levels increase cAMP levels, cAMP binds to CAP which causes a conformation change and allows DNA to bind to the CAP activator - no lactose: repressor binds (lac repressor) which blocks polymerase from binding - lacotse available: lactose is converted to allolactose which binds the lac repressor, DNA is unable to bind to the lac repressor allowing the lac operon to be expressed

DNA Polymerase Mechanism (and Active Site)

- active site shape drives nucleophilic attack by the 3'OH towards the alpha phosphate - uses single active site to catalyze all bases since A:T and C:G have similar shapes - prevents rNTP (precursor nucleotides) incorporation because rNTP cannot fit in the active site - also uses Mg2+ to promote attack by deprotonating 3'OH of primer and coordinating the negative charge of beta and gamma phosphates

Translocation (EF-G)

- after peptidyl transferase rxn, tRNA in P site is no longer attached to amino acid - for a new round of elongation, P site tRNA must move to E site, A site tRNA must move to P site, and the mRNA must move by 3 nucleotides - after peptidyle transferase rxn, factor binding site (A site) is uncovered and here EF-G GTP binding stimulates hydrolysis at A site - EF-G can only bind to ribosome when associated with GTP - GTP hydrolysis changes EF-G conformation so it can reach into the small subunit - EF-G drives location by displacing A site tRNA - EF-G GDP mimics that of tRNA bound to protein

Homologous Recombination Step 1

- alignment of 2 homolgous DNA molecules

Divergent Transcription at Promoters

- all RNA polymerase II promoters are not direction specific in mammals and will actually use the other strand - positive direction: splice sites in DNA, full length transcription - negative direction: RNA gets transcribed quickly with no splice sites and a shorter transcript, generally non functional

Mismatch Repair and Cancer

- all eukaryotic cells have proteins analogous to MutS/MutL (MSH2 in humans) - defects in MSH2/MLH1 predispose patients to familial colon, endometrial, and ovarian cancers - account for up to 15% of colorectal cancers!

Using Reverse Transcriptase

- all eukaryotic mRNA end in poly(A) - mRNA template of only T's: oligo dT primer that can base pair to all mRNA - reverse transcriptase and dNTP (used in PCR) yield complementary DNA which is a mRNA-DNA hybrid - mRNA is then degraded with alkali (base) - key uses in RT-PCR and recombinant DNA

RNA Viruses

- all require RNA-dependent RNA polymerase (RdRP) which is an early protein made with retroviruses that catalyzes RNA replication from RNA template, EXCEPTION TO CENTRAL DOGMA - single stranded can either be positive strand that is directly translated into protein or negative strand which can base pair with mRNA to make mRNA - double stranded can be A double helix or retrovirus

Cascade of Alternative Spicing: dsx (double sex)

- allows different splicing, but BOTH are functional - females: tra interacts with dsx so get one type of splicing - males: splice and get functional version but alternative splicing pattern because no tra

Topoisomerases are Drug Targets

- antibiotics (ex: quinalones target DNA gyrase) - cancer therapy (ex: campothecin analogs inhibit eukaryotic type I)

DNA Replication Rules

- applicable to all organisms 1. semiconservative: each strand is a template 2. begins at an origin of replication 3. proceeds bidirectionally 4. synthesis is semidiscontinous (replication fork with lagging and leading strands)

mRNA Degradation

- as soon as mRNA gets to the cytoplasm, degradation begins 1. way of recognizing mRNAs with mistakes (degrade faster) 2. another way to regulate gene expression (protein count)

Chemistry of Transcription (additional molecules that help)

- at 3' end: 3 Asp that stabilize/position 2 Mg2+ and these Mg interact with triphosphate that is next nucleotide to be added (nucleotide base based on base pairing of template) and 3' OH at end of RNA performs nucleophilic attack of alpha-phosphate with new phosphodiester bond - core principle of DNA/RNA polymerases

Chemistry of DNA Synthesis (Reaction)

- attacks alpha phosphate (phosphoryl group transfer rxn) - the products of the first reaction include PPi which is further broken down to make the reaction irreversible/favorable - PPi breakdown drives the reaction

Regulation of Transcription (levels of transcription)

- basal level of transcription: -35 and -10 are capable of doing - add repressors: no transcription, binds between -35 and -10 and prevents polymerase from binding - add activators: increase transcription, promotes transition from closed to open complex - activator protein: interacts with polymerase and isomerize DNA

Spliceosome: 5' Splice Site Recognition

- base pairing between U1 and 5' splice site - recruits U1 snRNP - U1 displaced by U6 snRNP (mediated by base pairing)

Spliceosome: U2:U6 Pairing

- base pairing can hold together snRNPs in spliceosome

Nucleosome

- basic structural unit of eukaryotic chromosome (beads on a string = 10nm fiber)

Insulator Elements

- binds regulator proteins: set up domains enhancers so they can't get through - insulator sets up domain with enhancers and promoters - barrier that enhancers can't get through to talk to the promoter

Detecting Methylation Genome-Wide

- bisulphite sequencing: specialized version of whole genome sequences - add bisulfate: methylated C stays same because it's protected by unmethylated C becomes a U

Phosphodiester Backbone

- bond formed between 5'-phosphate of one nucleotide and 3' hydroxyl of next - nucleic acid sequences are 5' to 3'

microRNA and RNA Interference Overlaps

- both are about 22 NTs long - both bind with Ago - extensive pairing between microRNA and target site (RARE) can result in cleavage - minimal pairing between single RNA and target site results in microRNA like repression

Cascade of Alternative Spicing: tra (transformer)

- both transcribe, SXL splices - females splice into tra - males are unable to get a functional protein

Spliceosome: Branch Point Recognition

- branchpoint binding protein (BBP) displaced by U2 snRNP - base pairing but this excludes A

Challenges to DNA Replication

- bumping into nucleosomes - nucleotide depletion - transcription intermediates - DNA-protein complexes - DNA lesions - double-strand breaks - secondary structures (G tetraplexes) - heterochromatin - transcription-replication collisions - replication-induced DNA damage can lead to oncogenes which result in genomic instability and increased proliferation of the oncogenes

Tryptophan Levels High

- can form C-G rich stem loop followed by Poly(U) - intrinsic terminator! (3:4 pair) where region 2 in ribosome doesn't pair, instead 3 pairs with 4 - synthesis stops, polymerase releases - made leader RNA, don't need to make Trp

Utility of RNA Interference Through Reverse Genetics

- can quickly inactivate a gene as longa s you deliver double stranded RNA - the hard part is getting RNA in - in c. elegans, you can simply feed the work a plasmid that expresses the double stranded DNA - in mammals, you need to transfect cells with a duplex of RNA - in humans, gene overexpression or a defective form can be fixed through RNA interference but it is difficult to deliver in vivo and target specificity is challenging

Initiation

- can start without primer (compared to DNA that needs a primer) - promoter recognition and binding - formation of closed complex (binding) - transition to open complex (breaking hydrogen bonds) - rounds of abortive transcription - promoter clearance (escape) where sigma dissociates - transition to elongation phase

Eukaryotic Cell Cycle

- cell cycle: series of events that takes place in a cell elading to its division and duplication 1. M phase (mitosis): chromosome segregation 2. Gap 1 (ends with 1 genome) 3. S phase (synthesis): DNA replication (ends with 2 genomes) 4. Gap 2

Protecting the Telomere

- cell may interpret presence of DNA end as a DNA break and attempts to repair this would result in chromosome fusion - proteins bind to the telomere to protect it - telomeres also form a t-loop to mask the end of it

Mechanisms of Alternative and Regulated Splicing: Activator

- cell type 1: no expression of splicing activator protein, no splicing - cell type 2: expression of splicing activator protein through binding the exon splicing enhancer to strengthen exon definition, results in splicing

Mechanisms of Alternative and Regulated Splicing: Repressor

- cell type 1: no expression of splicing repressor protein so splicing occurs - cell type 2: expression of splicing repressor protein and exon definition weakened so mRNA not spliced

DNA Supercoiling in Cells

- cells maintain DNA in supercoiled and underwound state to facilitate replication and transcription & compaction - allows access to this information - topological state dynamic and highly regulated since LK can be changed (not LK0)

Imprinting

- certain genes retain epigenetic marks and determine expression of gene in next generation (gene expression determiend by parental origin) - ex: methylation prevents enhancer from interacting with insulator and H19 because methyl groups are too dense across tehse regions - this is rare: ~100 imprinted genes in mammals

Genome Structure (Eukaryotes)

- chromosome: linear DNA molecule (23 pairs) with telomeres and centromere - unique sequences (genes) are in chromosomes - DNA is highly packaged, either as heterchromatin which is very dense and few genes or euchromatin which is less packed with many genes

Termination in Eukaryotes

- cleaving and releasing RNA is key event - first model: Rat1/hXrn2 is loaded onto Pol II and a poly(A) sequence directs to clip RNA and release it, Rat1/hXrn2 recognize uncapped RNA since the uncapped end is its substrate which degrades nucleotides until it catches Pol II and stops it (torpedo model) - second model: polymerase becomes less possessive and falls off DNA due to cleavage and release of RNA, reduces chance Pol II stays (allosteric model)

tRNA

- clover leaf model, all have CCA at 3' terminus after transcription - amino acid covalently attached - anticodon recognizes codon - 61 codons but fewer tRNAS

Genomics in Medicine

- ethical/legal/social issues resulting from sequencing: confidentiality of data, appropriate use of data, clinical issues where mutations influence probability of a disease - we have the tech to sequence a genome, the challenge is to understand the genome

Mediator Complex

- co-activator: take diverse inputs (activating and repressing) and signal to Pol II, or in other words integrate enhancer and transcription factor inputs - physically interacts with Pol II - lots of proteins in the mediator complex - first input: enhancers bound by transcription factors which physically interact with mediator - second input: chromatin modification that change interactions between subunits and DNA/enhancers (high mobility group non histone chromatin proteins contribute as well)

Structure of TADs

- cohesin: forms ring structures - CTCF: DNA binding protein, tethers DNA loops

Transcriptional Regulation

- cohesin: protein that tethers far away enhancers to the promoter - topologically associating domains (TADs)

Discovery of Splicing

- colinearity of gene and protein: line up amino acid sequence and proteins - splicing breaks up colinearity! - Roberts and Sharp worked with human viruses and cut DNA with restriction enzyme and then incubrated with heat. this allowed the DNA to be partially unwound and then RNA could base pair with DNA

New Generation of Anticancer Drugs

- combination approach - ex: cancer cell with a tolerance to replication stress evolved to have increased DNA repair, but becomes addicted to this tolerance mechanism so if we are able to identify the mechanism we can inhibit it as well as induce more replication stress

Eukaryotic RNA Polymerases

- common ancestor so similar structure, but eukaryotic polymerases have additional subunits and do NOT have sigma subunits - some viral polymerases are closer to DNA polymerases - 3 types of polymerase all eukaryotes have (plants also have pol IV and pol V) 1. pol I (specialized): ribosomal RNA 2. pol II: messenger RNA and more (most complex regulation because of the number of genes transcribed) 3. pol III (diverse): non-coding RNA

Histone Modifications & Histone Code

- common modifications: phosphorylation, methylation, acetylation, more - modifications are combinatorial to create a complex code: different combinations and modifications have different meanings - histones undergo multiple forms of covalent modification that impact chromatin through net charge, shape, etc. - ex: acetylation = remove postive charge

Anticancer Drugs Target DNA Replication

- common target to prevent tumor growth - targets: synthesis of dNTPs (5-FU), chain elongation (AraC), DNA template (Cisplatin), DNA polymerase (Aphidocolin) - however this is not very specific, has too many side effects, and can result in resistance by cancer cells

DNA Constantly Damaged

- common types: 1. depurination and deamination (cytosine transformed to uracil through hydrolysis) 2. oxidation: reactive oxygen species from metabolic processes 3. alkylation: chemicals (ex: N-nitroso compound in meat damages DNA) 4. pyrmidine dimers: UV light 5. mismatches: replication errors 6. double strand breaks: ionizing radiation, replication fork encountering lesions

Replisome

- complex of enzymes and proteins at the replication fork - enzymes: 1. helicase: separate strands 2. ligase: seal gaps of Okazaki fragments 3. topoisomerase: topological relief 4. polymerase: DNA synthesis

Cloning Vectors

- contain DNA of interest, allow for replication and purification - derived from plasmids (double stranded, circular, 20 kb) - important regions: 1. origin of replication 2. drug resistance marker (choose bacteria with plasmid) 3. multiple cloning site (recognition sequences for restriction endonucleases stitched together, more versatile)

Spontaneous Reversion of 10E-7 with 400 million cells. If a mutagenic compound is added that increases mutagenicity by 3 fold, how many colonies will be on the control plate and the compound plate? What if the bacteria is lacking mismatch repair, then how many revertants?

- control plate: 40 - compound plate: 120 - control plate without mismatch repair: 4,000

Movement of DNA and RNA During Elongation (Correcting Errors)

- correcting errors (wrong nucleotide) 1. pyrophosphorolytic editing: same active site, backward reaction because everything slows down 2. hydrolytic editing: enzyme backtracks, cleaves RNA with errors by using GRE factors which cleave wrong pair - NO exonucleolytic proofreading like DNA polymerases so RNA is more error prone than DNA - RNA consequences: wrong AA in protein compared to DNA consequences where it is permanent and all RNA is changed and it's inherited in a cell

Capping

- covalent modification of 5' of all Pol II mRNA - cap is a nucleotide attached by a 5'-5' triphosphate bridge and is added soon after transcription is initiated - added by RNA triphosphatase (makes bridge), guanyltransferase and methyltransferase (adds methyl) - roles for cap: 1. promote export (allows it to be exported out of nucleus) 2. stabilize RNA (protects it from degradation) 3. stimulate translation (tells cells to translate)

Alterations in Chromatin Again

- covalent modification to histone tails: acetylation of lysine to make it more transcriptionally active by changing to euchromatin - structural modifications (non covalent): chromatin remodeling complex recruited by transcription factors that changes position of DNA relative to histones so promoter can be accessed - many different histone modifications with 3 categories of enzymes 1. writers: covalently add groups to tails 2. erasers: remove acetyl groups, methyl groups, phosphates 3. readers: recognize individual covalent modifications and only bind if specific one is there (impact gene expression) - histone modifications can be complex (histone code) - ex: HP1 binds if a Me is there and this will condense chromatin, but if there's a phosphate at the next nucleotide then HP1 is forced to dissociate

Eukaryotic Gene Structure (mRNA): Recall

- create precursor mRNA, splice, then have mRNA - splicing begins as transcript is made

Generating Recombinant DNA

- cut DNA with recognition sequences (ie. with restriction endonuclease) - cut plasmid so compatible with DNA (base pair perfectly, use same restriction endonuclease) - mix together (sticky ends cause base pairing while blunt ends come together with base stacking, the cloning vector then has the DNA sequence in it as well) - add DNA ligase to fix phosphodiester backbone

Codon Usage

- degenerate codons not used equally - codons used frequently correspond to high level tRNAs - codon bias differs between species - codon bias can create problems for recombinant protein expression (if rare codons are used, it is difficult to efficiently translate mRNA)

EF-TU

- delivers aminoacyl-tRNA to A site (escorts) which increases the accuracy of translation - EF-TU is bound to GTP - many aminoacyl-tRNAs will enter but only the correct one stably binds and then EF-TU further stabilizes correct tRNA - EF-TU positions at factor binding center (above A site) after tRNA in A site is bound stably and then GTPase activity is activated when at the center if the amino acid is correct - GTP becomes hydrolyzed and EF-TU then has no affinity for the ribosome and leaves, leaving the tRNA behind - incorrect delivery of aminoacyl-tRNA accounts for most errors in translation

Fidelity via Base Pairing

- depends on geometry of active site and Watson-Crick base pairing - in vitro this alone results in 1 in 10E5 incorrect nucleotide added which means other mechanisms exist

Differences in Gene Expression Underlie...

- different cell types - disease (cancer caused by mutation in gene expression) - speciation (genes being expressed at different levels in different species) - differences in population (mutations cause different expression) - transcription controls gene expression

Pathways to Repair DNA

- different pathways deal with different types of damage - many pathways conserved from bacteria to humans

Timing of Origin Replication

- different regions replicate at distinct times in S phase to maintain genome stability - different times because of the amount of dNTPs (if all at once then would consume all the dNTPs and the replication forks would stall which is really bad because single stranded DNA can be cleaved and this can damage DNA) - having different times allows dNTPs to be made - some cancers allow all replication origins to fire at once - timing is influenced by local chromatin structure established during Gap 1

Alternative Splicing and Disease

- disease can arise because of alternative splicing = ex: cystic fibrosis

Homologous Recombination Step 6

- double holiday junctions can be formed and can result in chromosome breakage so holiday junctions need to be resolved - resolvases clip at a holiday junction to create chromosomes without crossover - resolvases can also clip above and below holiday junction to create chromosomes with crossover - RuvC in e. coli

Translation Initiation in Eukaryotes (5' dependent translation)

- driven by 5' cap which recruits initiation factors (5' cap dependent translation) - 48s = pre-initiation complex which positions first the Met by scanning for AUG and then 80s initiation complex - use start codon, initiator tRNA, initation factors

Ribozymes

- drives translation - can also make man made ribozymes that are capable of partial self replication - ex: hammerhead ribozyme can cleave RNA

Telomeres and Disease

- dysfunctions in telomerase in humans associated with disease

Charging tRNAs with Correct Amino Acid

- each 20 amino acid attached to tRNA by one tRNA synthetase - 1 enzyme:1amino acid:multiple tRNAs - challenge: match correct set of tRNAs for amino acid (frequency of mischarging: <0.005%) - how to ensure fidelity in tRNA charging: shape and size of amino acid binding site (but some amino acids very similar)

Termination

- elongation ends when stop codon enters the A site (UAA, UAG, UGA) - release factors recognize stop codons

Homologous Recombination Step 5

- elongation of invading strand to regenerate broken DNA - invading strands end in 3' so can be primers - properly repairs using information in the homologous chromosome (error free!)

Finding Start Sites

- emerging techniques of DNA sequencing to map start sites - ribosome profile: RNase 1 treatment to cause ribosomes to accumulate at start site, then isolate polysomes and digest the free mRNA and isolate the remaining mRNA to sequence - this reveals non-canonical start sites (synthesize in response to environment): used to regulate but don't know relevance

2nd Type of Alternative Splicing

- encode 2 proteins with 2 different functions

Regulation of Transcription in Eukaryotes

- enhancer can function 5' or 3' of promoter, can recruit activating or repressing factors, is independent of distance, and sequence can work independent of which strand of DNA it's on (more flexibility) - 50-100k enhancers(?) since many genes have multiple enhancers - transcription factors: often conserved across species, determine expression of body parts (ie. wing vs limb)

TADs

- enhancers can be folded to interact with promoters - only interact with promoters in ONE TAD - boundaries between TADs are insulators - TAD architecture is different in different cell types

DNA Synthesis in Vivo

- enzymes separate strands - strands only separate at the bubble - primers made by enzymes - need topoisomerase - replication forks exist - dNTP levels maintained by an enzyme - lower error rate (1 in 10E9) - whole genome replicated - Okazaki fragments occur

Synthesizing aminoacyl-tRNAs

- enzymes that attach correct amino acid to tRNAs are aminoacyl tRNA synthetases - two steps: adenylation and tRNA charging which uses one ATP

Topoisomerases

- enzymes that can regulate topological state by changing LK - can relax supercoiled DNA - can separate based on topological state where more supoercoiled travels faster because smaller volume - targets tense molecules in a step-wise fashion (increase LK by one or two)

Restriction Endonucleases

- enzymes that recognize specific sequence in DNA and cleave DNA within recognition sequence - considered bacterial immune system where they play a protective role that digest foreign DNA but host DNA is protected by site specific DNA methylase that interferes with function of enzyme (host DNA is modified) - restriction endonucleases and methylase have overlapping recognition sequences - binds dimer that is symmetrical and cleave and then leave a short single base ("sticky end") - 3 types: 1. make sticky ends at 5' 2. make sticky ends at 3' 3. make blunt end (not as useful)

Why do biological systems tolerate RNA polymerases with a far higher error rate than that of DNA polymerases?

- erroneous transcripts will just be turned over/recycled and at most, a handful of incorrect protein will be translated - for organisms, RNA is not the genetic material

mRNA Surveillance

- errors in mRNA synthesis/processing - trigger mRNA decay pathways

Differences in Translation Initiation Between Prokaryotes and Eukaryotes

- eukaryotes use Met while prokaryotes use fMet - eukaryotic small subunit binds initiator tRNA before mRNA recruitment while prokaryotic small subunit binds mRNA before initiator tRNA - eukaryotic start codon found by scanning downstream of 5' end - eukaryotic uses 5' cap - eukaryotic has a lot of proteins recruited

Chromatin (Regulation and Organization)

- eukaryotic chromosomes larger with higher degree of organization - chromatin = DNA + tightly bound proteins - chromatin is useful for organization: wrapping DNA around protein spools - chromatin is useful for regulation: expand how compaction can be regulated - histones represent major class of proteins in chromatin

Nucleosomes Store Negative Superhelicity

- eukaryotic topoisomerases cannot introduce negative supercoils - instead, get underwound DNA through histones - DNA binding to the histone core results in a negative supercoil spiral with a plectonemic region that is unbound and positive supercoil - topoisomerase relaxes the positive supercoil to result in one negative supercoil around the histone core

mRNA Export (Nucleus to Cytoplasm)

- export stimulated by: 5' cap, poly A tail, proteins deposited by splicing - nuclear pore complex sets up/governs export and import from and to the nucleus. this is regulated!

Base Pairing in RNA

- extra hydroxyl group on sugar means additional H bonding - U can pair with G as a wobble pair

SDS-PAGE

- extract proteins out in globular native form but there is some interaction through disulfide bonds that help with their tertiary form - treat proteins with SDS detergent to denature (and heat) - use mercaptoethanol to reduce disulfide bonds - proteins must be linear in order to infer molecular weight - every protein surrounded by SDS results in a negative charge where they can migrate to the positive cathode - small proteins travel faster and can run with standard proteins that you know the molecular weight of

Mechanism of Type 1

- first conformational change promotes strand passage - second conformational change brings cleaved ends together - model for Type II similar but invovles dimers (2 reactive Tyr)

Homologous Recombination Step 3

- formation of 3' tails with RecBCD (MRX in eukaryotes)

mTOR: Mammalian Taget of Rapamycin

- from streptomyces - discovered in 1975 by Rapa Nui - used as immunosuppressant to prevent organ transplantation rejection but it is now an anticancer drug

Genes

- gene is a region of genome that is transcribed, a sequence in DNA that instructs transcription - transcription takes one side into coding transcript (mRNA) and is then translated into a protein - can also become a non-coding transcript (rRNA, tRNA, etc.) - 3 characteristics of a gene: transcription, regulation of synthesis, molecule has some type of function

Homologous Recombination

- genetic exchange between pair of homologous DNA sequences - homologous sequences: DNA sequences similar or identical in NT's (>50 bp) - DNA breaks are required to initiate recombination

Transcriptional Control by Promoter Strength and Sigma Factors

- globally changing many gene's transcription levels simultaneously - ex: heat shock allows sigma 32 to take over and transcribe

Supercoiling in Eukaryotes

- have topologically restrained regions - cannot freely rotate along its lengths - organized into loops whose ends are restrained

DNA Synthesis in Vitro

- heat separates the strands - strands become fully separated - primers are made synthetically and added - no topoisomerase - no replication forks - add in dNTPs - higher error rate (1 in 10E5) - small region amplified - no Okazaki fragments

Unwinding Double Stranded DNA

- helicase is enzyme that further unwinds double stranded DNA to expose single stranded DNA - helicase is ring shaped, 6 subunit complex that: 1. encircles single stranded DNA in lagging stand 2. uses ATP hydrolysis to move processively in the 5' to 3' direction (in eukaryotes encircles the leading strand and moves 3' to 5' direction)

DNA Binding at lac operon (protein shape)

- helix turn helix is protein shape that binds DNA and is found in CAP and lac repressor - helix turn helix binds at half sites of lac operator (repeating sequence)

RNA Seq

- high sequencing to mRNA detection and quantification - isolate mRNA with poly A tail (bead attached to oligo dT to fish out mRNA) - then fragment DNA with reverse transcriptase and primer and add all possible DNA sequences as primers - convert DNA so it can be sequenced - then map to gene! lots of sequences at exons, not introns

Alterations in Chromatin

- histone acetylation changes heterochromatin to euchromatin (more open so TFIID can bind easier) - done by adding acetyl group to Lys on the tail

Octamer, Contacts, Tails

- histone core has two copies of each histone to make an octomer - DNA circumnavigates more than once and makes extensive connections (14 contact sites, one contact site occurs every 10.5 turns) - histone tails protrude out and can have post translational modifications

Histones (Classes and Amino Acid Composition)

- histone proteins have 5 classes in all eukaryotes (conserved) - H2A, H2B, H3, & H4 create the core histone - H1 is for linker DNA - histones are very basic with lots of Lys and Arg (20% Lys/Arg) - if Lys residues replaced with Glu, modified histone would have less affinity for DNA because Lys is positively charged

Nucleotide Excision Repair and Cancer

- human genetic disease Xp: defect in enzyme for py-dimer excision that leads to skin cancers - extreme cases: exposure to sunlight forbidden (ie. midnight children who can only play outside at night)

Biological Roles for microRNAs in Mammals

- humans have hundreds of microRNAs - each microRNA represses many different mRNAs - many mRNAs repressed by microRNAs - repression sites mainly 3'UTR - pairing to 5' end of microRNA (seed) is most important

Guanosine Tetraplex

- hydrogen bonding in G tetraplexes allows them to become stacked - can be found in RNA and DNA - highly stable due to stacking - can interfere with behavior of synthetic DNA/RNA structures

Chromatin Immunoprecipitation Assays (ChIP)

- identify all DNA sites bound by protein of interest - take chromatin and cross link (covalent links between DNA and proteins that are reversible) - fish out transcription factor of interest by using an antibody that binds specific proteins (protein of interest) - immunoprecipitation: purify protein of interest - reverse crosslinks, remove protein - then sequence with illumina

Modern Sanger Sequencing

- improvement: fluorescent terminating groups that are not radioactive and can look at color instead so each base is a different color and only one rxn is needed - incorporation of the dideoxy terminates growth and labels DNA fluorescently and then detector can read with electrophoresis - still need primer and DNA polymerase

Formation of Supercoiling

- in B form of DNA, one helical turn = strands wound around each other one time (once every 10.5 base pairs) - supercoiling forms when 2 strands of circular DNA duplex wound around each other - ex: 84 base pairs: 8 turns would be relaxed (stable) while 7 turns would be underwound (unstable, triggers negative supercoil) and 9 turns would be overwound (unstable, triggers positive supercoil)

How to Tell Which Strands Contains the Mistake?

- in E. coli,. Dam methylase methylates A resides on both strands of a 5'-GATC-3' sequence - these sequences can be 1000 base pairs away - in daughter strand though, GATC is not methylated yet - MutH binds at the hemimethylated site and activated when bound by MutL/MutS and it only nicks unmethylated strands - unclear how this happens in eukaryotes (maybe sliding clamp?)

Exon Definition Model

- in humans and other complex models, exons are small - splice sites are short and occur in "wrong" place - splicing machinery finds introns through the exon definition model - here we need extra specificity through exon splice enhancers: extra sequence information in exons recognized by SR proteins before splicing - if RNA not bound by SR, then it's an intron

If a gene coding for an mRNA were engineered to be expressed by RNA polymerase I, what three problems might you expect to occur during synthesis of this mRNA

- in order for Pol I to transcribe the mRNA, a promoter to recruit pol I must be incorporated - the level of complexity of the Pol I promoter is vastly different than that of pol II - no distal regulatory enhancers associated with promoter - RNA pol I primarily produces a single transcript while Pol II must transcribe a variety of transcripts - transcription by Pol I would not be as efficient, with a lower precision

Why Eukaryotic Chromosomes are Replicated Once per Cell Cycle

- incomplete replication: causes inappropriate links between daughter chromosomes and separating these linked chromosomes causes breakage/loss - re-replication: leads to amplification of specific regions which can lead to catastrophic defects/disease (also called copy-number variation and many cancers take advantage of this)

Fidelity via Exonuclease Proofreading

- incorporation of incorrect nucleotide slows DNA synthesis which results in the dNTP not being held against the template well - the exonuclease active site has 10 fold higher affinity for a single stranded 3'OH - once in the active site, the incorrect nucleotide is cleaved - this is 3' to 5' econuclease activitiy

Intron Definition Model

- information that drives splicing - don't need anything extra to recruit splicing machinery - how splicing works in yeast: small genome, strong splice sites

Homologous Recombination Step 2

- introduction of breaks in DNA

RNA Interference (RNAi)

- introduction of double stranded RNA in c. elegans inhibits expression of genes with the same sequence as RNA - inhibition is highly potent - purified single strand RNA is ineffective at inhibiting

Allostery

- lac repressor is dimer, nestled into major groove w/o allolactose so w/ allolactose induces conformation change and cannot bind to DNA - add cAMP to CAP: forms H-bonds with amino acid side chains that stabilize extended helical region which twists recognition helices and can nestle into major groove

Organization of DNA: TADs

- large regions with many genes - roles for CTCF and cohesin - promoter-enhancer interactions within TADs - can be repressed

Ribosome Composition

- large unit and small unit - prokaryote: large unit is 50s and small unit is 30s with overall unit as 70s - eukaryote: large unit is 60s and small unit is 40s with 80s overall - eukaryotic and prokaryotic slightly different so opportunity for antibodies - named according to velocity of sedimentation under centrifugation

BRCA1 Mutations

- leads to breast cancer - 55-60% with the mutation develop breast cancer by 70 - BRCA1 acts at step 3 with the 3' tail formation - eukaryotes have other ligases that can just join blunt ends however these can incorrectly fuse so they are very error prone and promote non homologus end joining (NHEJ) - BRCA1 mutant cells instability is induced by deregulation of NHEJ

Protein Synthesis More or Less Complex than DNA Replication/Transcription

- less complex: does not have to be as accurate, 200-1000 nucleotides per sec - more complex: no specificity affinity between amino acids and bases, polymerization of 20 amino acids, only 2-20 amino acids per sec, protein synthesis can account for up to 90% of chemical energy used by cell

Proteomics: Why Study Proteins?

- level of transcription does not correlate well with protein abundance (high mRNA level but translation can be stopped) - cell diversity generated by post-transcriptional regulation (includes post-translation modifications) - post translation modifications and protein-protein interactions cannot be inferred from transcription profiling - protein: effector molecules - protein malfunction associated with disease

Post Translational Modifications

- lots of modificaitons (most proteins modified by covalent attachment of molecules to amino acid - ex: phosphorylation, acetylation, lipidation, hydroxylation, SUMOylation, glycosylation, disulfide bond, ubiquitination, methylation, and more (400+ found) - post translational modifications regulate a range of protein properties (structure, interactions, activity, locations, functions)

If Kronberg used gamma-P32 instead in his experiments would it have worked to discover the first DNA polyemrase?

- no because the radioactive dNTP would not be incorporated since the alpha phosphate is what is incorporated

Ribosome

- macromolecular machine that coordinates mRNA/tRNA recognition (decoding) and the formation of a peptide bond (peptidyl transferase)

Major and Minor Grooves

- major are wider/deeper while minor are narrower/shallower - chemical identity of side chains of hydrogen bonding changes in major/minor grooves - there is a unique code that distinguishes pairs in the major groove - less information in the minor groove

DNA Methylation

- majority of methlaytion on CpG (C is methylated) - >50% CpG methylated in most cells - methylation represses transcription when there's lots of CpGs as a CpG island - if active transcription, not methylated - methylated CpG island causes inactive genes so a CpG that is methylated but NOT an island is NOT repressive

trp operon

- makes Trp - trp repressor regulated by Tryptophan with Trp repressors (repressor binds DNA) - low Trp: synthesize operon - med Trp: terminate transcription at attenuation sequence

Sex Determination

- males with one X chromosome express sisterless-a and sisterless-b which are transcriptional activators encoded on the X chromosome and they also express deadpan, a transcriptional repressor encoded on an autosome (no transcription) - females have 2 X chromosomes so express MORE sisterless-a and sisterless-B and this allows them to transcribe SXL

Why are changes to the genetic code so rare?

- many amino acids can be coded by multiple codons so one letter changes might not change the amino acid, this gives our DNA more robustness - changes to the genetic code can be disastorous with severe consequences - if a change were to occur such as one amino acid codon being changed, this would impact every protein in cells which can affect protein structures and functions - an exception would be changes to the mitochondrial genome since its genome is very small

Sensor Kinases Promising Targets for Drugs

- many kinases rely on these for tolerance mechanisms 1. ATR 2. ATM 3. DNA-PKcs

Internal Ribosome Entry Sites (IRES) (5' cap independent)

- mechanism of alternative initiation: 5' cap independent - here the mRNA mimics protein and the normal requirements for translation initiation are bypassed (bypass elF4E or initiator tRNA and directly assemble) - three reasons why this may exist: 1. viruses enter and cells shut down 5' dependent translation to stop the virus but the virus evolved to bypass this 2. mitosis shuts down 5' dependent translation but if we need a protein we can bypass 3. this can occur when 5' dependent is inhibited

Telomeres and Aging

- mice without telomerase have premature aging - if telomerase is overexpressed, cells are immortalized

Repression by microRNAs

- microRNA + Ago = RISC - Ago can speed up deadenylation, so it degrades mRNA and repressed the gene

Regulatory RNAs

- microRNA, RNA interact - long intergenic RNA - CRISPR RNA

Organelle Genomes

- mitochondria: present in ~all eukaryotes, densely packed and circular DNA - chloroplast: present in plants - endosymbiotic theory: eukaryotic mitochondria originated as bacteria which was engulfed by another cell

Transposons

- mobile elements: segments of DNA that can move within the genome - considered selfish since they can copy themselves and copy their copying mechanism - present in many copies per genome - not all copies are functional - some evidence that they contribute to gene regulation - many different types/mechanisms - in both eukaryotes and bacteria - contribute to insertions, deletions, translocations - major source of variation in genome sizes due to transposons

Histone Code

- modifications focused on promoter - covalent modification changes where you are in transcribed genes (don't fully understand) - not fully stable (can be reversed) - ex: telomeric: end of histone, little modification - ex: centromere: hyperacylated - ex: active and repressed euchromatin

Nucleic Acid Double Helices

- monomers can exist in different configurations - A form: dehydrated samples, typical for RNA double helix - B form: all DNA in nature - Z form: CG repeat sequences, very rare in nature

Nucleotides

- monomers of nucleic acid - bases are glycosidically bonded to sugar - bases are bonded to phosphate group with ester bond

Dscam Alternative Splciing

- most alternatively spliced genes in Drosophila - many alternatives to each exon: choose one type of exon and can have 38,000 variant proteins - involved with neural development

Mouse microRNAs

- mouse microRNA knockouts show that some are essential and other create severe phenotypes

Helicase Activation

- need polymerases and kinases (CDK and DDK) - NOT in bacteria - phosphorylate helicase directly - phosphorylation converts assembled pre-RC to activated helicase

Post Translational Events

- needs to mature into functional protein by folding events 1. fold into unique 3D conformations 2. bind cofactors 3. covalent modifications: post-translational modifications 4. assemble into large multi-subunit complexes

Recombinant DNA

- new DNA sequence created by combining existing sequences - cuts phosphodiester bond in a staggered break

Double Strand Break Sources

- nicks, collapsed replication fork - incomplete replicated DNA (mitosis) - ionizing radiation - transposable elements

tRNA Introns Splicing

- specific to tRNAs - mechanism: cleavage and ligation - catalytic machinery: endonuclease + ligase

RNA Pol II Carboxy Terminal Domain (Tail)

- no phosphorylation pre-initation - during promoter escape, second Ser becomes phosphorylated to recruit capping factors - during elongation, first Ser is phosphorylated and second Ser is not to recruit splicing factors - poly A factors also on tail because of phosphorylation

Histone Modifications at Yeast Telomere

- no transcription over telomeres 1. Rap1 recognizes sequence 2. Rap2 recruites Sir 3. Sir2 is eraser, deacetlates proximal nucleosomes 4. deacetylated nucleosomes bind more Sir (Sir3 and Sir4) 5. Sir3 and Sir4 erasers: deacetylate over long ranges, results in spreading of silenced domain

DNA Methylation and Gene Repression in Development

- normal development: accumulation of cell type specific methylation patterns (repression) - ageing: methylation patterns change with age, loss of methylation typically - cancer: changes in methylation (gain and loss) - methylation results in proteins that bind methylated DNA being recruited which then recruit more repression proteins - fertilized zygote has methylation but removed as matures to blastocyst which has no methylation and then blastocyst maturing to embryo does have methylation - also primordial germ cells have methylation but it is removed as it matures into sperm or egg so the sperm or egg get their own methylation pattern - TAKEAWAY: remove and reset to get rid of methylation and then accumulate again

Termination

- not well understood - two pathways: 1. intrinsic (rho independent): need certain sequence with symmetry in DNA that will fold into a hairpin and also have a run of U's. if this forms, then will release RNA and NO extra factors are needed 2. rho-dependent: ATP-dependent helicase recognizes rut (rho utilization) sequence that is rich in C and rho binds nascent transcript and migrates towards polymerase where interactions between RNA polymerase and rho result in termination

30 nm Fiber (Formation)

- nucleosomes can be packed on top of another to generate a second level of of chromatin that is 40 fold compact and called 30nm fiber - formation of 30nm requires: one molecule of H1 per nucleosome which changes DNA curvature and brings histone cores close and interactions between tails of adjacent nucleosomes (modify tail changes interaction and enzymes can then change from 30nm to 10nm and back) - 30nm fiber is associated with regions of DNA that aren't transcribed

Substrates Required by DNA Polymerase

- nucleotides, template strand of single strand, primer - add nucleotides to 3'OH but cannot start new strand so primer offers the 3'OH as the template junction

Initiation Mechanisms

- observed: 5-10 nucleotide RNA and then dissociates: abortive RNA, polymerase tries again - models: 1. transient excursions: moves down DNA but sigma still in contact 2. inchworming model: entire complex doesn't move, only part moves down and other part stays 3. scrunching model: polymerase stays, DNA extruded out as like a loop (most likely model to be true)

Example of Combination Therapy

- onco gene that has deregulated DNA replication and replication stress which causes DNA breaks and cell death - cell uses ATR to increase DNA repair and prevent DNA breaks - anticancer drugs to induce replication stress and inhibit ATR

Oncogenes Impair Proper Regulation

- oncogene-induced replication stress - oncogenes increase proliferation capacity of cancer cells while imparing proper control of origin firing (overfiring, refiring, asymmetric firing) - transcription-replication collision can result in double stranded break or depletion of replication factors/dNTPs - these collisions result in genomic instability

Estimate how many mis-incorporated amino acids are expected to be present in all protein molecules present in one nucleosome core

- one nucleosome = 8 histone proteins - one histone protein = 120 amino acids - one nucleosome therefore has ~1000 amino acids - error rate is 0.1% so 0.001*1000 = 1 amino acid

Elongation

- only core enzyme, no sigma - NusA binds: elongation factor that facilitates efficient elongation, leaves at end of transcription - sigma cycle: terminate transcription, core enzyme can find new promoter and start again

Viral RNA

- only one DNA strand to generate RNA: big regulation step

Polymerase Chain Reaction

- only sequence of interest cloned - heat denatured (98ºC) to get single stranded DNA - add primers to flank region you want to amplify - cool down so primers will base pair - add DNA polymerase from thermophyllic bacteria - then repeat to clone more

Polymerase I

- only transcribes ribosomal RNA precursor (ribosomal RNA makes up majority of all RNA) - huge need and multiple genes encode ribosomal RNA - has a upstream control element and a core promoter where transcription starts - proteins latch on: UBF (upstream binding factor) binds to the UCE (upstream control element) and SL1 (selective factor 1) binds to promoter and includes TBP in it (TATA binding protein)

DNA as Genetic Material: Experiments

- people thought genetic material was protein based since DNA was too simple - experiment one: found that isolated DNA from pathogenic cell added to nonpathogenic cell resulted in DNA converting to pathogenic state and changed the cell (not proof because DNA not perfectly pure) - experiment two: worked with phage and separately labeled DNA and protein and then added to bacteria and blended and it was shown that the protein didn't enter the bacteria but the DNA did and it was sufficient to allow the phage to replicate

Cyclin Dependent Kinases (CDK) Regulate DNA

- phosphorylates many proteins to regulate processes - low CDK activity during G1: allows pre-RC formation - high CDK activity during S phase: allows existing pre-RCs to be activated (not all origins are used though) and these pre-RCs are disassembled after being activated or the DNA is replicated - during each cycle, pre-RCs can only be formed once and can only be activated once

Plasmids

- plasmids typical in bacteria (easy to engineer, invade cells, medical relevance) - plasmids are small (few kb, <10 genes) and are autonomous mobile genetic elements: 1. one bacterium contains plasmid to be transferred 2. connection forms and plasmid is copied 3. both bacteria now contain the plasmid

Transcription and Translation in Bacteria

- same components in cell (not same in eukaryotes since transcription is in nucleus and translation in cytoplasm) - can associate with ribosomes: transcribed and translated at same time

Cleavage and Polyadenylation

- poly(A) sequence (AAUAAA) in RNA that is a binding site for CstF (cleavage stimulation factor) and CPSF (cleavage and polyadenylation specificity factor) - move from tail to poly(A) 1. cleave RNA 2. CstF dissociates 3. CPSF recruits poly A polymerase (doesn't use template) 4. poly A polymerase adds lots of A's 5. poly A tail substrate for PABP (poly A binding protein) - roles (similar to cap): 1. promote export from nucleus to cytoplasm (only capped/tailed RNA is exported) 2. stabilize RNA from RNAses 3. stimulate translation - ultimately end up with poly A sequence bound by PABP

Completing Lagging Strand Synthesis

- polymerase I removes RNA primers (5' to 3' exonuclease proofreading) and replaced with DNA (5' to 3' polymerase) - DNA ligases seal the nicks with a phosphodiester bond between adjacent 5' phosphate and 3'OH - RNase H can also remove RNA primers (which is why polymerase I is not essential)

DNA Polymerases are Processive

- porcessivity: average number of nucleotides added each time the enzyme binds the primer template junction - polymerase holds onto the subtrate and continues the cycle for many nucleotides - processivity relies on the ability of polymerases to slide along DNA template by using the "thumb" of the "hand" to hold DNA when the "fingers" open

Patterns of Transcriptional Regulation

- positive regulation: bound activator facilitates transcription (cAMP and CAP) - negative regulation: bound repressor inhibits transcription (allolactose and lac repressor or trp + trp repressor)

Initiating DNA Replication (polymerase switching too)

- pre-RC activation = helicase activation - depends on kinases CDK and DDK - phosphorylation of helicase and other proteins leads to helicase activation and stepwise recruitment: 1. DNA polymerase epsilon and DNA polymerase delta (processive polymerases) 2. DNA polymerase alpha (primase) which makes the primers but has low processivity 3. sliding clamp & clamp loader (initiate processive DNA synthesis) - polymerase switching: after initial synthesis, DNA polymerase alpha (primase) is rapidly replaced by the more processive polymerases (epsilon for leading strand and delta for lagging strand)

Alternative Splicing and its Significance

- pre-mRNA can be spliced in more than one way - can result in alternative functions but many alternatives are not functional - significance: 1. efficiency: only one gene needed and can swap splicing 2. regulation***: can regulate gene expression with functional and nonfunctional splice forms 3. diversity: diversifies the protein

mTOR Inhibitors in Translation

- prevent mTOR activity which prevents phosphorylation of 4E-BP - having more 4E-BP that is able to inhibit results in less translation - less translation = less growth

Why Use Erasable RNA Primer?

- primase is more error prone but synthesizing RNA means it's easy to know what to erase when it's time to remove the primer

Making Primers

- primase is specialized RNA polymerase dedicated to making short RNA primers - primers are synthesized in the 5' to 3' direction on single stranded DNA and are 5-10 nucleotides - primers have low processivity and low fidelity - the leading strand has one primer - the lagging strand has multiple primers - polymerase III extends primers to generate 1,000 nucleotide fragments - DNA can use RNA or DNA primers

End Replication Problem

- primer removed so in lagging strand 3' tail exists at the end of the chromosome - lagging strand synthesis is unable to copy the end so the chromosome becomes shorter - telomeres prevent this problem by creating a buffer that contains simple repeat sequences (up to 1000 times of short TG rich sequences called tandem repeats)

DNA Replication

- primer strand: DNA polymerase need already double stranded piece that has a 3' OH at the end - DNA polymerase chooses correct nucleotide and covalently attaches using the 3' OH to base pair and then repeats

Translation Initiation in Bacteria

- prokaryotic mRNAs recruited to small subunit by base pairing to the ribosomal RNA at the P site (ribosome binding site with 16s rRNA) - initiator tRNA charged with modified Met (fMet) at the start codon in P site - initiation factors direct assembly of initiation complex

Bacterial Gene Structure (mRNA and regulation)

- promoter: directs transcription to mRNA then translates to protein - on sides of coding sequences: untranslated region (5'UTR and 3'UTR) - 5'UTR recruits to regulate translations

GTP Binding/Hydrolyzing Proteins Coordinate Translation

- protein conformation changes depending on GDP or GTP binding - GTP hydrolysis used to monitor completion of key steps (ex: EF-TU only hydrolyze GTP after correct base pairing) - each step is interdependent and is a good antibiotic target because stop one part results in the whole thing stopping (ie. one tRNA gets stopped)

Protein-DNA Binding

- protein nestles into major groove - non covalent interaction between side chains of proteins and side chains of bases - ex: Zinc finger domain, homeodomain

Western Blotting (Immunoblotting)

- proteins first separated by SDS page - then proteins are transferred to hydrophobic membrane where they are immobilized - antibodies are used to detect protein of interest (more antibodies = more protein) - most widely used technique to quantify expression levels - only useful to study known protein whose antibody is available

Chemistry of Protein Synthesis: in Vivo vs in Vitro

- proteins: long polymers of amino acids linked by peptide bonds - in vitro, small proteins chemically synthesized but low efficiency and max of 100 amino acids in a few days - in vivo, some proteins have 4000+ amino acids and 100 amino acids can be synthesized in 5 seconds, amino acids added to growing polypeptide chain, tRNAs match amino acids to codons in mRNA AND drive rxn by activating the carboxyl group (peptidyl transferase rxn helps remove the OH group)

Base Composition of DNA

- purine content = pyrmidine content - therefore: adenine content = thymine content and guanine content = cytosine conent - base compositions differ between species - within a species, all tissues have the same base composition - bases have tautomoers (non-dominant vs dominant forms) but they are stable in one tautomer because of side chains

Visualizing Replication of E. Coli

- see a bubble with replication forks - replication only in 5' to 3' direction (DNA polymerase can only add in that direction) - use 3H to visualize

Incorporation Experiments with Isotopes to Study DNA Synthesis

- radioactive isotopes: signal detected using films that is incorporated into part of the nucleotide that will be retained in final DNA (32P, 3H) - stable isotopes: DNA becomes heavier and can be separated from normal DNA (2H, 15N)

Rate of Origin

- rapidly dividing cells have small replicons - slowly dividing cells have large replicons - rate regulated by number of initiation sites activated

Group I Introns Splicing

- rare, nuclear RNA, in some eukaryotes - mechanisms: two transesterification reactions and branch site G - catalytic machinery: ribozyme

21st Amino Acid

- rare, select proteins that use 21st amino acid: selenocysteine - in 3'UTR there is a SECIS in some RNA (weird bump looking thing) - SECIS changes the identity of the stop codon UGA and becomes selenocysteine instead with special tRNA that recognizes UGA but is charged with Ser that is converted to Sec - downstream of UGA is UAG which is the stop codon used

Group II Introns Splicing

- rare, some genes from organelles (mitochondria, chloroplast), prokaryotic - very old, origin of splicing since it can self splice, likely evolved into spliceosome - mechanism: two transesterification reactions and branch site A - catalytic machinery: RNA enzyme encoded by intron (ribozyme)

Regulation of Translation

- rate of translation responsive to several factors since it is a huge energetic cost to the cell if it isn't regulated - ex: external stimuli such as nutrient availability where low nutrients reduces translation and high nutrients increases translation

Mutations Disrupting TADs

- rearrange genome results in different borders of TADS - this leads to misexpression

DNA Recombination

- recombination: process in which DNA molecules are broken and fragments are rejoined in new combinates - generates genetic diversity during meiosis - regulation of gene expression

NtrC and glnA

- regulate nitrogen levels and uses sigma 54 - NtrC = regulatory protein, activates transcription, exception is function for up to 2kb from promoter that is unusual for bacteria - IHF bends DNA, brings NtrC closer to polymerase to allow polymerase to activate

Genetic Code

- relationship between bases in DNA and resulting protein that is synthesized - almost completely universal - evolved a long time ago

Bacteria Replication

- replicate DNA all the time - origins of replication may fire constantly - not much regulation

Eukaryotes Replication

- replicate DNA only once per cell cycle at the S phase - origins of replication fire once per cell cycle - extensive regulation

Fidelity

- replication error rate is only one per billion (10E9) - 3 steps for high fidelity 1. base pairing through 5' to 3' polymerization 2. error correction through 3' to 5' nuclease activitiy 3. error correction through strand directed mismatch repair

Prokaryotes Overall

- replication fork geometry: same as eukaryotes - core enzymes at replisome: DNA primase, RNA primer, DNA ligase, DNA polymerase alpha and beta, helicase - genome size: 1-10 million base pairs - chromosome structure: circular - speed of replication fork movement: 1,000 nucleotides per second with one origin of replication

Eukaryotes Overall

- replication fork geometry: same as prokaryotes - core enzymes at replisome: DNA primase, RNA primer, DNA ligase, DNA polymerase alpha and beta, helicase and more - genome size: 10 million base pairs-3,000+ million base pairs - chromosome structure: linear with telomeres - speed of replication fork movement: 50 nucleotides per second with multiple origins of replication (much slower because nucleosomes are obstacles in the way, 33 days to replicate human chromosome)

DNA Replication and Topological Problem

- replication requires separation of DNA strands which requires the rotation of rest of DNA double helix and that creates a positive supercoil - topoisomerases essential for DNA replication by traveling in front of areas of strand separation and allow rotation of short length of helix to relieve tension

DNA Polymerase Structure and Cycle

- resembles right hand - cycle: 1. correct base pairing 2. "fingers" close 3. nucleophilic attack 4. "fingers" open 5. movement of primer template junction by one base pair

Advantage of Controlling Protein Synthesis vs. mRNA Transcription?

- respond faster to making proteins since already have the mRNA - much faster to break and release the break (more dynamic, rapid response) - translational control happens at initiation mostly (don't stop synthesis halfway)

Ribosome Structure & Function and Ribosomal RNA

- ribosomal RNA responsible for ribosome's overall structure, ability of ribosomes to position tRNAs on mRNA, and catalytic activiting in forming peptide bonds - large subunit: contains peptidyl transferase center - small subunit: contains decoding center

Tryptophan Levels Low

- ribosome gets stalled because next two codons code Trp - slows down, not lots of tRNA for Trp - when stuck, very long structure that is stable is formed (2:3 pair) so the attentuator is not formed and it fully synthesizes the mRNA - eventually tRNA gets there and adds Trp - ribosome disrupts structure and transcribes to eventually make more Trp

Translation Elongation (What Happens, Elongation Factors)

- ribosome has initiator tRNA 1. second aminoacyl-tRNA enters the A site 2. peptidyl transferase reaction occurs (forms peptide bond) 3. A site tRNA translocated to P site, P site tRNA moved to E site - then it's ready for another charged tRNA - elongation highly conserved between prokaryotes and eukaryotes but they have different elongation factors - bacteria: EF-TU, EF-G - eukaryotes: EF1, EF2

Myriad microRNAs

- roles for small RNAs in plants - also found in let-7 c. elegans and other animals (highly conserved) - animals and plant genomes encode hundreds of microRNAs

Unknown Protein Identity?

- run SDS page but can't do a western blot so instead do mass spectroscopy - mass spectroscopy most sensitive and fastest technique to identify proteins - cut up protein and throw in to get fingerprint of peice - interrogate genome database to get sequence and identify the protein - mass spectroscopy determines the mass with high accuracy and sensitivity

Gel Electrophoresis of Nucleic Acids

- separation of nucleic acids by application of voltage difference across gel matrix - charge on nucleic acids results in mobility within electric field (from neg to pos) - uniform structure of linear nucleic acids results in fractionization based on size (longer move slower)

Splice Sites

- sequences in pre-mRNA - exons have conventional bonds while introns have lariat strcuture - 3 sequence elements: 1. 5' splice site: GU (first intron nucleotides) 2. 3' splice cite: AG (last intron nucleotides) 3. branchsite A

Testing Possible Models (Meselon-Stahl Experiment)

- set up: E. coli grown in 15N for many generations and then grown in 14N for one generation - put samples into a CsCl solution in a centrifuge and distributive and semiconservative result in mixed sample while conservative stays separate - grow E. coli for a second generation in 14N and centrifuge again - distributive stays completely mixed, semiconservative is partially mixed partially new, and conservative is completely separated still

LINC RNA Example: Xist RNA

- sets up equivalent expression (dosage compensation) - female mammals have 2 X chromosomes but one X is inactive - male mammals only have 1 X chromosome - Xist binds one X, recruits polycomb, and silences the chromosome so it will not be transcriped - therefore, LINC RNAs recruit chromatin modifying proteins to specific locations in the genome

Sigma Factors (E. coli)

- sigma binds promoter to initialize transcription (controls which promoter) - most bacteria have multiple different sigma factors (E. coli has 7 sigma factors) - sigma 70 is normal sigma factor in E. coli that synthesizes the most genes - sigma 32 is how heat shock genes are synthesized - sigma binds to promoter, transcribes early phage genes and then one early phage gene makes sigma factor with higher affinity and replaces sigma 70 which transcribed middle phage genes and it repeats to transcribe late phage genes

Promoters in Bacteria

- sigma recognizes -35 and -10 region - also up element that is an additional sequence present some promoters and interacts with one alpha subunit - not all promoters match perfectly - promoters that match well are strong promoters (housekeeping genes) because those genes are needed often (up element means stronger promoter because more contact) - promoters that deviate are weak promoters since those genes are only needed in low levels (ie. the nucleotides don't match or spacing is not exact) - promoter very different are recognized by different sigma and can regulate large groups of genes differently

Typical RNA Structures

- single strand that can fold back on itself - form a hairpin - form a tetraloop (end of loop of hairpin) - form a base triple (U:A:U, additional motifs that stabilize) - pseudoknots: single strand with 4 regions that can base pair to each other can forms two loops where distant regions fold up and stabilize further - kissing loops: distant parts come together where the loops at ends base pair to each other

Meiosis Favors Inter-Homolog Recombination

- sister chromatids avoid each other and go to non sister inter-homolog instead - resolution step will determine if homologous recombination will lead to gene conversion or cross over - however homologous chromosomes have a similar learn array of genes (one bp different in one million bps)

Types of Alternative Splicing

- skip an exon - exon extended into intron sequence - intron retained - alternative exons

Sliding Clamp

- sliding clamps opened and placed by clamp loaders (once for leading, multiple for lagging) - to load: use clamp loader enzyme with lots of ATP - loads a clamp each second on the lagging strand - clamp loader coordinates both strands simultaneously

Variations in Mitochondria Genetic Code

- slight changes to genetic code in mitochondria genomes - mitochondria only encodes small number of proteins though (compared to if it was in the nucleus it would change a lot) - these slight changes happen with stop codons

Okazaki Fragments

- small fragments formed in the lagging strand of DNA that eventually become ligated together to form a full strand - Okazaki grew E. coli exposed to 3H for 15 sec and extracted the DNA to find 50% large molecules and 50% small molecules (2000 nucleotides or so) - then put in a 5 minute chase and extracted DNA to find majority large molecules

Mutations in Code

- some mutations won't change encoded amino acid sequences - synonymous mutations: no change in encoding - non-synonymous mutations: change in encoding - frame shift mutation: insertions/deletions (that aren't multiples of 3 nucleotides) will compeltely alter the encoded amino acids 3' of the mutation - mutations that do not change amino acid sequence can have an effect but this is RARE - 2 levels: 1. AA unchanged bc same AA with different codon 2. AA changed to similar AA

Editing Pocket Increases Fidelity

- some use editing pocket - Ile synthetase has editing pocket near synthesis site that proofreads aminoacyl-tRNA products - Val-tRNA can enter the editing pocket because its smaller where it becomes hydrolyzed meaning it is the incorrect amino acid - Ile-tRNA too big so doesn't become hydrolyzed

microRNA Overall

- source: endogenous gene - targets: many - repression: subtle - mechanism: inhibition of translation - target pairing: minimal (~8bp)

RNA Interference Overall

- source: exogenous gene - targets: one - repression: variable - mechanism: mRNA cleavage - target pairing: extensive

Meiosis

- special cell division that generates haploids - resulting chromosomes in haploid cells is unique mixture of original homologous chromosomes - leads to gene conversion and cross overs - for genetic diversity, recombination CANNOT occur between sister chromatids

Prokaryotes with Special Topoisomerase That Introduces Supercoils

- special type II: DNA gyrase which is a good antibiotic target - DNA gyrase keeps bacterial genome in negative supercoiled state by introducing negative supercoils and relaxing positive supercoils - eukaryotic topoisomerases cannot unwind DNA so instead DNA binding proteins introduce negative supercoiling

Discovery of First microRNA

- special way of triggering decay - found mutant c. elegans lin-4 gene that produced a developmental defect - when they mapped where the mutation occurs, region of mutation did not contain a protein coding gene - discovered function of lin-4 is to repress lin-14 - lin-4 gene product is a small, non-coding RNA which represses lin-14 by pairing with a sequence in the 3'UTR of lin-14

Chromosomes with Multiple Origins of Replication

- speed of fork movement in eukaryotes slower with a bigger genome - eukaryotes have multiple origins of replication (10,000 origins separated by 30kilobases) - not all origins are needed to complete the genome - allows for more regulation (timing/rate of origin usage used to dictate replication rate)

Cascade of Alternative Spicing: SXL (sex lethal)

- splice into mRNA if have SXL - maintain production of SXL in females - males unable to splice because didn't transcribe SXL so they have a nonfunctional protein

Epigenetics (and mechanisms)

- stable change in gene expression without change in DNA sequence - epigenetic decision to determine eventual fate of cell (permanent change in gene expression but DNA sequence is unchanged) - mechanisms of epigenetic events: 1. DNA methylation 2. Histone modification

Homologous Recombination Step 4

- strand invasion establishes base pairing between DNA molecules - initiates exchange of DNA strands - heteroduplex is formation that checks sequence homology (3' tail of one chromosome pairs with strand of other chromosome) - holiday junction is the crossing of DNA strands for invasion - RecA assembles on single stranded DNA to form nucleoproteins and extends length of DNA by 1.5 fold (adjacent bases 5 angstroms apart) and promotes invasion (Rad51 in eukaryotes) by mediating search and catalyzes pairing

DNA Polymerase I

- structural gene: polA - processivity: 3-200 base pairs - polymerization rate: 16-20 nucleotides per second - subunits: 1 - 3' to 5' exonuclease proofreading: Yes - 5' to 3' exonuclease proofreading: Yes

DNA Polymerase II

- structural gene: polB - processivity: 1,500 base pairs - polymerization rate: 40 nucleotides per second - subunits: 7 - 3' to 5' exonuclease proofreading: Yes - 5' to 3' exonuclease proofreading: No

DNA Polymerase III

- structural gene: polC - processivity: >500,000 base pairs - polymerization rate: 250-1,000 nucleotides per second (double for the 2 replication forks) - subunits: 10 - 3' to 5' exonuclease proofreading: Yes - 5' to 3' exonuclease proofreading: No

Implications of Structure for Replication of DNA

- structure independent of sequence, but sequence specific recognition of DNA is essential - both strands can be replicated due to base pairing

Pairing in RNA

- subtle differences in RNA and DNA - thymine replaced by uracil allows for wobble pairing - G can pair with C or U - U can pair with A or G - I (inosine) can pair with C, U, or A - therefore an anticodon can recognize multiple codons - this interaction only occurs in the ribosome which holds the tRNA and wobble pairs only occur at the far right position (the wobble position) in the ribosome

High-Throughput Sequencing

- suite of alternative sequencing (next generation sequencing) - most common: Illumina (much faster and cheaper) - core tech for genomics

DNA Supercoiling

- supercoiling: coiling of a coil, distortion caused by torsional stress - result of topological property of DNA - only applied if both strands remain covalently intact and tension is stored

Local Histone Modifications by Polycomb in Animals

- switches off gene 1. PRE (polycomb response elements): DNA sequence triggers repression 2. PRE recognized and bound by PHO-RC 3. PHO-RC recruits PRC2 (polycomb repressive complex 2) 4. PCR2 is a writer: contains histone methyltransferase to generate a methylated histone 5. PRC2 and methylated histone recruit PRC1 which is a reader and represses through nucleosome positioning and chromatin condensation

Complex RNA Structures

- tRNA: unusual noncovalent interactions between nucleotides - rRNA (ribosomal RNA, drives translation and called ribozyme): core component of translation machinery, EXCEPTION TO CENTRAL DOGMA - adopt 3D structures that are very stable and molecule can be catalytic (act as enzymes!)

Telomerase (Function and Composition)

- telomerase extends 3' end of telomere so an additional 3' end DNA can act as a template for a new Okazaki fragment however the 3' overhand still exists - telomerase is a ribonucleo protein containing RNA where RNA is a template for the telomeric sequence repeat (reverse transcriptase)

Inhibitors of Protein Synthesis as Antibiotics

- tetracycline targets A site of 30s and inhibits aminoacyl-tRNA from binding to the A site which isn't a problem for eukaryotes because it looks for a specific form only in prokaryotes - 2/3 of antibiotics come from streptomyces (broad spectrum antibiotics) - paromycin binds to codon-anticodon site in 30s and increases rate of error of translation - chloraphenicol blocks correct positioning of A site so peptidyl transferase rxn doesn't occur

What would happen in Reiji Okazaki's classical experiment if he quickly inactivated sliding clamp loaders during the "pulse" period? Explain what the results would be, and how they would be different compared to what Okazaki observed in his classical experiment.

- there would be no large fragments because the DNA polymerase is not processive enough

Linking Number

- topological property, used to measure degree of supercoiling - LK = number of times one DNA strand winds around the other - LK0 = number of base pairs/10.5 (in B form, relaxed state) - only applied if both strands covalently intact - twist = # of turns, writhe = # of supercoils - LK = twist + writhe (stays the same if not cut so decrease writhe = increase twists and vice versa) - ex: 2100 base pairs, LK0 = 2100/10.5 = 200 - ex: 84 base pairs with 7 turns: must have one negative super coil to have 7 twists so writhes = -1 because 7 turns is underwound and need to get to 8 turns but to get to 8 turns must adjust by the writhes which is -1 - ex: 5250 base pairs with 4 writhes, how many twists? LK0 = 500, twists = LK0 - writhe therefore twists = 496 = LK

DNA Topology

- topology: spatial properties that are preserved under continuous deformations of objects

Eukaryotic Gene Structure (mRNA and regulation)

- transcribed unit with promoter and enhancer (these together drive transcription) - often many enhancers for one promoter, can be 100,000 nucleotides away from the promoter - initially synthesized precursor mRNA that has introns, which are regions that get removed through splicing and the exons (regions that get left behind) are stitched together to make mRNA - this is NOT colinearity: mRNA without introns is colinear - final mRNA also has a coding sequence, 5'UTR and 3'UTR - however the sequence is majority intron

Polymerase III

- transcribes variety of noncoding RNAS: 5sRNA, tRNA, more - has box A and box B where TFIIIB and TBP bind along with TFIIIC (TF = transcription factor) - recognition sites after initiation site get transcribed

How Are Genes Regulated?

- transcription - processing (splicing) - export - decay - microRNAs - translation

Activator Proteins (2 domains)

- transcription factor with 2 domains: 1. DNA binding domain (sequence specificity) 2. activation domain (signals to mediator) - experiment: - replace Gal1 with lac z because lac z will turn blue when expressed and then link Gal4 with lac z - when Gal4 is expressed, lac z will turn blue - if we remove the activation domain, lac z is not turned on - if we use lex A instead of Gal4 for DNA binding domain it's not on - if we fuse DNA binding of lex A with activation domain of Gal 4 it does turn on - therefore: transcription factor for DNA binding and activation are separate

microRNAs and Cancer

- tumor suppressors

Cooperativity and Combinatorial Regulation

- two enhancers are next to each other: transcription factors interact and can get multiple bases on enhancer or protein-protein interaction (cooperative recruitment) - in specific chromatin state only one enhancer active: the enhancer is a remodeler and can modify so the other enhancer becomes active (indriect effects) and the enhancer can also recruit chromatin remodeling complex - SWI5 binds and recruits remodling complexes (exposed SBF to turn gene on)

Chromatin

- two major functions: packaging and regulation - DNA wrapped around histone to make nucleosome, then nucleosomes are wrapped again to become very packed - 2m long can fit into a nucleus - enhancers are active in euchromatin only

Topoisomerase Types

- type 1: change LK by 1, cleave one strand, no ATP needed - type 2: change LK by 2, cleave both strands, require ATP

Nucleotide Excision Repair

- type of lesion: "bulky" lesions that cause helical structure distortions (pyrmidine dimers by UV light)

Base Excision Repair

- type of lesion: modified bases - uses glycosylases which are abundant and fast (first line of defense) - this also works if a base is missing

Mismatch Repair

- type of lesion: replication errors that escape proofreading - mismatch repair detects and repairs these lesions quickly and before the second round of replication - increases fidelity by 100 fold

Translesion Synthesis

- type of lesion: replication fork encountering damaged DNA that can't base pair so it can't be a template - here DNA pol III falls off and Pol IV and Pol V (Y family polymerases) come in but they are much more error prone since they synthesize DNA without base pairing (bypasses lesions) - however this is because incomplete replication is much worse

RNA Polymerase

- unwinding DNA to make bubble of single stranded DNA - multiprotein complex with 2 alpha subunits, one beta, one beta prime, one omega, and sigma - beta: polyermase activity itself - beta prime: binds DNA - alpha: assembly, interact with regulatory factors, catalysis - omega: doesn't know what it does - sigma: brings polymerase to promoter and helps open the DNA helix - shaped like crab claw - core properties same throughout all organisms

Most Carcinogens Cause Mutations

- use Ames test to measure carcinogenicity with specialized bacterial strain that cannot synthesize histidine - a mutagenic chemical will revert the frameshift mutation to allow cells to grow on media without histidine - more mutagenic chemical = more bacterial colonies formed - some colonies will form on control plate because of spontaneous revertants through errors

Amount of DNA in Nucleosome

- use nuclease with different digestion - light digestion only cuts through linker DNA (many bands throughout the gel) whereas extensive digestion cuts all DNA not on the histone (only one band towards the bottom) - highly conserved: always 146 base pairs wrapped around histone core - linker changes species to species (in humans one nucleosome = 200 base pairs) - procedure: 1. use nuclease to digest accessible DNA 2. extract DNA fragment from nucleosome core using high salt 3. run DNA fragment with gel electrophoresis 4. electrophoresis reveals the size of the fragment

Original Data Leading to Discovery of Spicing

- used electron microscopy - DNA much bigger than region RNA was pairing with - the excess regions were introns - this means there is discontinuity where introns must be taken out

Illumina Sequencing

- uses chain terminating nucleotide (no OH, color coated) - can record the color and cleave the group and continue to add nucleotides - sequencing in parallel many DNA molecules is much faster and cheaper - fluorescent dye is covalently attached at 3' position that can be removed and replaced with a 3' OH

Spliceosome

- very big: 5 snRNAs (small nuclear RNAs): U1, U2, U4, U5, U6 - 100 proteins (+100 accessory and regulatory factors) - snRNA + protein: snRNP complex (small nuclear ribonucleo protein) - has 5' splice site recognition, branch point recognition, and U2:U6 pairing

Nuclear pre-mRNA Splicing

- very common, most eukaryotic genes - mechanism: two transesterification reactions and branch site A - catalytic machinery: major and minor spliceosomes

DNA Sequencing: Maxam Gilbert

- very complicated and toxic 1. radio label 5' end with p32 2. cleave single stranded molecules @ specific base 3. run on gel, only read radioactive ones 4. repeat: G then C then A then T (A and T less specific)

Bacterial Operon

- very rare in eukaryotes - operon: multiple coding regions regulated with one promoter - genes with related functions often clustered in operons, this facilitates coordinate regulation of the operon - single polycistronic transcript (mRNA) with multiple coding sequences: individual proteins are translated

Solving Structure of DNA

- x-ray diffraction of DNA fibers - determined: helical molecule, 20A diameter, 3.4A repeat (turn), 36A repeat (nucleotide unit), antiparallel, right handed, double helix, major and minor grooves - Watson and Crick determined specific pairing of bases (A+T and C+G) - A+T has two hydrogen bonds whereas C+G has 3 hydrogen bonds and is more stable

If a circular bacterial chromosome exists as B-form DNA and is made of 2,100 base pairs, how many twists of the double helix would you expect in a relaxed DNA molecule? How would gyrase affect the linking number of the relaxed molecule if ten enzymatic reactions were allowed to take place? What is the new linking number? If five positive supercoils were introduced into the relaxed DNA, what is the new linking number?

1. 200 twists expected 2. new LK = 180 (10 enzymatic reactions would introduce 10 supercoils so LK would go down but because it's DNA gyrase it's a type II so it goes down by 2 each time) 3. new LK = 205

Three Binding Sites for tRNA (and channels)

1. A site: binds aminoacylted tRNA 2. P site: binds peptidyl-tRNA 3. E site: binds exiting tRNA that's released - channels: allow mRNA and polypeptide chain to enter/exit the ribosome

Three Key Features of Watson-Crick Base Pairing

1. A+T and G+C 2. AT and GC pairs are equivalent in size and shape 3. hydrogen bonds are an integral part of stability

Base Excision Repair Mechanism

1. DNA glycosylase cleaves n-glycosyl bond creating AP site 2. AP endonuclease cuts phosphodiester DNA strand with AP site 3. DNA polymerase I and DNA ligase repair the gap

Three key pieces of data that helped solve the structure of DNA

1. Knowledge of Base tautomers: a predominant stable arrangement of base side chains (or H bond Acceptors / donors) 2. Chargaffs rules: reciprocal relationships between A and T, and C and G - that is A content = T content, etc. 3. X-ray diffraction data, gave physical parameters that DNA must obey, e.g, helical.

Modifications of Histone Tails

1. Lys acetylation increases accessibility - acetyl-transferase (writers)that acetylates come close and modified histone to have a decreased affinity for DNA so it impacts its ability to form 30nm fiber - also recruites nucleosome remodeling complexes such as the bromo domain that recognizes the acetylated tail 2. histone deacetylases (erasers) removes acetyl groups

Four Types of Splicing

1. Nuclear pre-mRNA 2. Group II introns 3. Group I introns 4. tRNA introns

Differences between RNA Pol I and RNA Pol II

1. RNA Pol I synthesizes only non-coding RNAs, whereas RNA Pol II synthesizes coding RNAs (& some non-coding) 2. Transcriptional regulatory sequences are all promoter proximal for RNA Pol I; enhancers (RNA Pol II) can be distal 3. RNA Pol II (&not RNA Pol I) functions with mediator (protein complex that integrates regulatory information)

Different types of regulatory sequences that can regulate the expression of eukaryotic genes transcribed by RNA polymerase II?

1. TATA box recognized by TBP and help pre-initiation complex formation 2. enhancer sequences bind to repressor or activator proteins and influence transcription 3. insulator elements prevent enhancer elements from affecting the wrong promoter

Pol II Core Promoter and GTFS

1. TBP: binds TATA 2. TFIIA: stabilizes binding 3. TFIIB: recruits Pol II-TFIIF complex 4. TFIIE: recruits TFIIH 5. TFIIF: binds to Pol II and TFIIB, prevents Pol II from binding outside of promoters 6. TFIIH: unwinds DNA, contains CTD-kinase that adds phosphates to Pol II tail 7. NELF and DSIF: pausing factors 8. P-TEFb: pause release factor

Homologous Recombination Steps

1. alignment 2. introduce breaks 3. formation of 3' tails (5' resection) 4. strand invasion 5. DNA synthesis/ligation 6. cleavage of holiday junctions

Differences between bacterial and eukaryotic polymerases?

1. bacterial polymerase has a sigma factor 2. eukaryotic polymerase II is highly regulated with many different transcription factors 3. eukaryotes have different polymerases 4. eukaryotes synthesize a pre-mRNA 5. the DNA being transcribed is packed by histones in eukaryotes 6. translation and transcription can occur at the same time in bacteria

Mechanisms to Select Against Incorrect Aminoacyl-tRNAs

1. base pairing: 16s RNA forms series of H bonds with codon-anticodon pair 2. enzymatic activity: GTPase activity of EF-TU is sensitive to correct base pairing 3. positioning and time: after EF-TU release, incorrectly matched tRNAs dissociate more rapidly

Events that can regulate the expression of eukaryotic genes transcribed by RNA polymerase II?

1. binding of a repressor to an enhancer inhibits the function of a nearby activator (competition or inhibition) 2. activator or repressor binds to enhancer element and interacts with the mediator (direct regulation) 3. histone tails become acetylated leading to euchromatin formation and increased accessibility of the promoter (increased transcription) 4. histone tails become deacetylated leading to heterochromatin formation and decreased accessibility of promoter (decreased transcription)

mRNA Degradation Steps

1. chew up poly A tail (deadenylation), if it's faster it's less stable mRNA 2. once most gone, enzymatic removal of the CAP by DCP2 clipping it off (can also occur at 3' end if exosome chews from 3' end) 3. quickly degraded with XRN1 in 5' to 3' decay

Topoisomerase Process

1. cleavage of DNA 2. passage of segment of DNA through break 3. reseal DNA break

Repressor Protein Mechanisms

1. competition: activator and repressor sites are close together, repressor can outcompete or hinder activator from binding 2. inhibition: repressor interacts with activation domain of activator and stops it talking to mediator 3. direct repression: repressor interacts with mediator 4. indirect repression: control histone state, recruit to de-acetylate histones and make the chromatin more compact - ex: Gal1 can have Mig1 repressor that recruits Tup1 to change chromatin

DNA Damage and Cancer

1. defects in repair pathway scan lead to cancer (ex: mutations in BRCA1/BRCA2) 2. chemicals that are mutagenic can lead to cancer (carcinogens)

3 Possible Mechanisms for DNA Replication

1. distributed: chop into pieces, each piece is a template, then put it all back together 2. semiconservative: each strand is a template 3. conservative: don't separate strands, whole thing is a template

Temporal Order in Activation Origins

1. early origins: initiated early every cell cycle (large bubble) 2. late origins: initiated late every cell cycle (small bubble, chromatin makes it inaccessible) - if early and late are removed from their native chromosomal location, the origins would replicate at the same time

Transcriptional Regulatory Circuits

1. feed forward amplification 2. bistable mutual exclusion 3. feedback oscillator

Mismatch Repair: Scanning, Nicking, Repairing

1. find mismatch: in E. coli, dimer MutS protein scans genome for distortion caused by mismatch 2. making nick: MutS bound to mismatch recruits MutL which recruits MutH to nick the strand (up to 1,000 bp away from mismatch) 3. repairing mismatch: regio nof DNA between nick and mismatch removed by exonuclease and single strand gap filled in by DNA pol III

Preparing for DNA Replication

1. formation of pre-replication complex (pre-RC) 2. origin recognition complex (ORC) is initiator protein complex which binds origin of replication and recruits remaining replication proteins and helicase loading proteins 3. Mcm2-Mcm7 is hexameric helicase complex and is not active when recruited so it is not able to unwind DNA or recruit polymerases yet - different from bacteria where binding of initiator (DnaA) is coupled to unwinding and polymerase recruitment

Why Sequence?

1. gene finding 2. insights into evolution 3. identification of functional sequences 4. finding mutations that cause/contribute to human health 5. biotechnology 6. agriculture

3 ATPases important for replication?

1. helicase: unwinds strands 2. clamp loader: increase processivity by loading the sliding clamp 3. topoisomerase type II: relaxes positive supercoils using ATP

Noncovalent Interactions in DNA

1. hydrogen bonds between bases: help stabilize the double stranded helix 2. repulsion between phosphate groups (they are quite negative): minimized by the location of phosphates on the exterior side of the double helix 3. base stacking (most important for stability of structure since base stacking occurs only within a double helix): helps stabilize the helix

Nucleotide Excision Repair Mechanisms

1. in E.coli: UvrA and UvrB scan genome for distortions 2. UvrB melts DNA to form single strand bubble around lesion 3. UvrB recruits UvrC nuclease: 2 nicks around the lesion ,creates a 12 NT single strand 4. UvrD helicase removes single strand DNA, DNA pol and ligase fill gap

Contrast canonical translation initiation in bacteria and eukaryotes. Your answer should outline at least 5 differences.

1. in bacteria, initial tRNA has fMet / In eukaryotes, is Met 2. in bacteria, mRNA binds small subunit first / eukaryotes, initial tRNA binds small subunit first 3. in eukaryotes, mRNA has 5' CAP/ bacteria do not have 5' cap 4. in eukaryotes, start codon is found via scanning / in bacteria is by direct positioning 5. in eukaryotes, many proteins bind and process mRNA before it engages the ribosome

Transcription Cycle

1. initiation: promoter recognition, transition from closed to open state, promoter escape (synthesis of 10 nucleotides to full elongation mode) 2. elongation: RNA synthesis, template unwinding/rewinding 3. termination: transcript release

Ribosome Cycle and Polysome (Prokaryotes)

1. initiation: small subunit binds mRNA 2. elongation: large subunit binds and aminoacyl-tRNAs enter 3. termination: entire complex dissasembles - in one cycle undergo association/disassociation - polysome: ribosome only can synthesize from one mRNA but each mRNA can be translated by multiple ribosomes

microRNA Biogenesis

1. long primary microRNA transcript: RNA pol II transcript 2. 1st processing step in nucleus by RNA endonuclease Drosha (cuts to get pre-microRNA) 3. pre-microRNA exported from the nucleus 4. 2nd processing step in cytoplasm by Dicer (pair of small base paired microRNAs) which removes loop 5. microRNA binds Argonaute (Ago) to create RNA-induced silencing complex (RISC)

Primary Components of Translational Machinery

1. mRNAs: information 2. tRNAs: physical interface 3. aminoacyl-tRNA synthetase: couple tRNA to amino acids 4. ribosome: machine that coordinates recognition and catalyzes peptide bond

Polymerase II Cycle

1. pol II general transcription factors that bring pol to promoter and help initiate transcription 2. pre-initiate complex ("closed complex") 3. open complex 4. initiation 5. promoter escape 6. elongation 7. termination

How can bacterial transcription be regulated?

1. presence of sigma factors 2. heat shock in E. coli (sigma 32 binds to the promoter of heat shock gene to promote expression) 3. regulatory binding sites are very close to the promoter 4. activator proteins that facilitate transcription 5. repressor proteins that inhibit transcription

Splicing Mechanism and RNA

1. recognition of branch point, 5', 3' splice sites by branchpoint binding protein (BBP) and factors 2. BBP replaced with recruited U2 at the branch point 3. recruitment of tri-snRNP: U4, U5, U6) and accessory factors leave 4. complex rearrangement: U1 replaced 5. catalysis: 1st transesterification reaction, U4 replaced 6. catalysis: 2nd transesterification reaction - happens while RNA is being made (cotranscriptional processing)

Meiosis Steps

1. s phase to duplicate DNA 2. meiosis I where genetic diversity is introduced 3. meiosis II where 4 haploid cells are generated

How to Distinguish Plasmids with and without a sequence for recombinant plasmids

1. sequence the insert 2. PCR across the insertion position 3. restriction endonuclease (where you will get the original plasmid and the sequence if it is there, otherwise you just get the original plasmid)

RNA Interference Mechanism

1. single RNA generation: recognized by dicer which slices into two single strands 2. single RNA and Ago targets mRNA for cleavage and cuts both in half ("slicing") 3. RNA has no tail or cap which results in rapid degradation

Types of Recombination

1. site specific recombination: requires defined sequence motif 2. homologous recombination: between identical or almost identical sequences 3. transposons: DNA sequences that jump @ specific sequences or randomly

Gene Regulation Cascade in Drosophila Sex Determination

1. transcriptional regulation: males do not have SXL protein while females do have SXL protein 2. alternative splicing regulation: males have a non productive splicing while females have productive splicing 3. alternative splicing regulation: both males and females splice an isoform of dsx 4. transcriptional regulation

Proteomics: Major Questions

1. which protein (identification) 2. which level of expression (quantification) 3. which post translation modifications 4. interacts with which proteins

Central Dogma

DNA -> RNA -> Protein

Adenine

Purine

Guanine

Purine

Uracil

Pyrimidine

Cytoseine

Pyrmidine

Thymine

Pyrmidine

Bases

purines: adenine, guanine pyrimidines: cytosine, thymine, uracil