Lecture 1:

• Operon: genes involved in a single pathway which determines the expression of the tightly regulated genes

  • Gene cluster and promoter at minimum

• Promoter sequences 

  • -35 box and -10 box and purine start site are most common in E. Coli

  • -35 box - TG box

    • Major binding site for sigma 70 (E. Coli sigma factor)

    • Consensus = TTGACA

  • -10 box - Pribnow box

    • AT rich → easier to unwind due to double bonds instead of triple bonds between nucleotides

    • Consensus → TATAAT

    • Major binding site  

  • Purine start site (A or G)

• DNA footprinting

  • The empty place is where the promoter region is bound to on gels

    • how it happens? → specific binding protein that recognizes promoter sites → protein binds to the promoter regions → DNAse (involved in footprinting) cannot cut where the protein is bound so there is no DNA in this region → electrophoresis helps us visualize the area where the DNAse cannot cut

• RNA polymerase

  • 1 in prokaryotes

  • 3’ -OH attacks 5’ triphosphate (nucelophilic attack)

  • 2 mg2+ cofactors

  • No need for RNA primer (unlike DNA polymerase) (will be a T/F question)

  • Coding Strand (sense) same as mRNA

  • Template is used with RNA poly to make mRNA and is opposite of the sequence

• RNA poly subunits

  • Sigma - recognizes promoter sequences

    • ESSENTIAL for promoter recognition

    • Does not bind to promoter DNA on it’s own

    • Recognizes promoters for housekeeping genes

  • Beta prime - Binds DNA

  • Beta - Binds NTPs and interacts with sigma, polymerizes RNA (Polymerase activity)

  • Alpha - essential for assembly and elongation

  • Omega - functionality and stability

    • One more thing to note is (alpha)2(beta)(beta’) is the “core enzyme”

    • The core enzuyme + sigma cofactor is the “holoenzyme”

• Initiation of transcription

  • Phase 1, binding - interaction between promoter and RNA pol

    • Formation of closed cmplx where DNA is not unwound → but then unwinds at the -10 to +2/+3

  • Phase 2, initiation - transcription initiation/promoter clearance.

    • 8/9 nucleotides initially synthesized → sigma subunit released → pol leaves promoter and elongates RNA

  • The “scrunching” model: DNA is pulled into RNA polymerase

  • Rifampicin binds (beta) subunit & blocks initiation

• Transcription Elongation

  • Highest speed among the 3: [DNA replication (is correct)], RNA transcription, Protein transcription?

  • Direction of transcription is 5’-3’ → 3’ end is correlated to positive supercoils(unwinding) and 5’ end is correlated to negative supercoils(rewinding)

  • RNA Polymerase is relatively accurate (1 err / 10000 bps)

    • Errors are okay, half life of mRNA is short, messages are degraded

  • Slower in GC rich areas (due to triple bonds)

  • Topoisomerases relieve supercoiling

  • Higher stability during elongation than initiation cmplx

  • 14bp melted to form transcription bubble

  • 8-9 nucleotides within bubble paired with RNA chain

  • Double stranded DNA opens up in front of the bubble and closes up as RNA polymerase moves along → transcription bubble extends from -12 to +2

• Core RNA polymerase

  • N-terminal domains of the alpha subunits allow them to form dimers

  • Alpha subunit N-terminal domains bind to the Beta/Beta’ subunits

  • Beta/Beta’ subunits interact extensively with one another and together form an internal channel and the catalytic site

  • Beta subunit has polymerase activity

• RNA polymerase does not move at a steady rate

  • Temporarily delayed at pause sites

  • Pausing may lead to arrest/termination

  • Arrest is important in proofreading

• GreB helps rescue an arrested complex (stalled)

• Transcription Termination

  • Rho-independent

    • GC-rich inverted repeat allows RNA to form stem loop -> reaches to within 7-9 nucleotides of the 3’ end of the RNA

    • U-rich stretch immediately after stem loop causes pausing and release

  • Rho-Dependent termination

    • Hexameric(6 rho factors) ATP dependent helicase, Rho-factor

    • Rho-factor releases RNA from RNA-DNA hybrid

    • Binds to RNA to disrupt RNA-DNA interactions

• TRANSCRIPTION VS DNA REPLICATION

  • Similarities

    • DNA is a template 

    • Synthesis of Complementary strand

    • Same mechanism of phosphodiester bond formation

  • Differences

    • Transcription is selective

    • 1 strand is used as a template for transcription

    • !!!!Transcription does not require a primer!!!! (most important)

    • Transcription is more error prone (no exonuclease activity)

Lecture 2:

• Why regulate gene expression?

  • Environmental factors: food sources change

  • Developmental/Differentiation

  • Cell specialization

• Regulation

  • Controlling transcription initiation is most common → most efficient to regulate at beginning of pathway

  • Recall → sigma factor → specific to promoter sequences → sigma factor itself is transcribed to enhance gene expression in certain genes → regulates how much of a gene is transcribed

  • DNA binding proteins

    • regulators → bind to specific sites that affect how RNA polymerase binds

    • Blocking sites → inhibitor 

    • Affinity for polymerase → activator

    • Some systems are regulated by both negative and positive controllers 

  • Modes for negative control

    • Effector causes dissociation of repressor from DNA 

    • Effector causes binding of repressor to DNA

    • Negative control bc it inhibits transcription (repressor)

  • Modes for positive control

    • Effector causes dissociation of of activator from DNA inhibiting transcription

    • Effector causes binding of activator from DNA, inducing transcription

    • Positive control bc activates transcription (activator)

• The Lac Operon: physiological background

  • Lactose degraded by beta-galactosidase (Beta-gal)

    • Degrades but also isomerizes into allolactose

  • Galactoside permease for uptake of lactose

• Operon Advantages/Disadvantages

  • Advantages:

    • Coordinate regulation of multiple genes using a single cis acting DNA site

  • Disadvantages

    • Individual regulation of transcription cannot occur

• Lac Operon

  • Both positive and negative regulators

  • Bound repressor inhibits transcription

    • Absence of lactose: repressor binds

  • Bound activator facilitates transcription

    • Presence of lactose: activator binds/prevents repressor binding

  • Encodes 4 proteins: 1 regulatory, 3 enzymes

  • Operon in order

    • Promoter I (P_I)

    • LacI: regulatory

    • PromoterL (P_L)

    • OperatorL: regulator binding site (O_L)

    • LacZ: B-gal (if knocked out no growth)

    • LacY: permease-transport (if knocked out no growth)

    • LacA: galactoside transacetylase

      • if the knockouts are combined then there will be growth bc of the diffusable protein products

  • Activity increases when lactose is exclusively present with no glucose

  • No lactose: repressor (I) active, binds operator, inhibits RNAP to make Z,Y,A

  • Allolactose is a side product, and an inducer (IPTG is also an inducer) (de-represses the reperssor)

  • Lactose present: inducer made → allolactose → binding of inducer to repressor → repressor does not bind operon

  • 3 operators

    • -82 (O3) and +412 (O2) are auxiliary, +11 is main operator (O1)

  • Lac repression binds either O3 or O2 and always binds O1 along with either

    • 4 LacI is nessecary to inhibit → dimerization at O1, and dimerization at O2/O3 → Dimers form another dimer → DNA loop

  • Lac operator is a palindrome → lacI is a dimeric protein and palindromic sequences are recognized by dimeric proteins

  • Lac repression involves DNA looping 

• Genetic perspective on lac operon:

  • Create lac- mutuants → lose a function or create a mechanism?

  • Complementation analysis 

Lecture 3:

• Cis vs Trans gene regulation

  • Cis acting elements → DNA sequences in vicinity of genes

  • Trans acting factors → diffusible protein factors that bind to DNA sequences

  • Note that cis/trans do not illustrate the bond formation

• Regulatory mutants

  • i- → mutant nonfunctional repressor protein

  • I^S → super repressor: cannot be inactivated by inducer

  • O^C → operator constitutive: cannot be bound by repressor

  • p- → nonfunctional promoter

• CAP protein & Cap site → glucose sensitive switch

  • Levels of ec glucose and intracellular cAMP → inversely related

  • High glucose levels outside cell create low cAMP levels within cell

  • CAP/cAMP = gene activator

  • CAP = CRP = “cAMP receptor protein”

    • Bind DNA when activated by cAMP

    • Binds lac operon promoter for lacZYA

• Note that Lac Operon is ONLY active under Lactose=present and glucose=absent conditions

• Catabolic vs Anabolic operons

  • Lac operon encodes catabolic enzymes

    • Catabolic operons generally regulated through induction

  • Trp operon encodes anabolic enzymes

    • Anabolic operons generally regulated through repression

• Operons involved in AA synthesis are tightly regulated

  • AAs are expensive

  • Aa levels are low → then expressed

  • Repressed when abundant

• Tryptophan Operon:

  • 2 modes of regulation

    • Trp repression

    • Transcription attenuation

  • Operon Order

    • trpP (promoter)

    • trpO (operator)

    • trpL

    • trpE

    • trpD

    • trpC

  • Repression:

    • trpR requires trp as a corepressor → tryptophan levels low → no corepressor → repressor does not bind to aporepressor → no binding to operator → no repression → Expression of trp operon

    • trpR + trp(levels are high) → trp binds to aporepressor → conformational change in aporepressor → binds operator → inhibits expression

  • !!!!Attenuation!!!!

    • Fine-tuning of synthesis

    • Low trp → full mRNA made 

    • High trp → leader sequence made (trpL) exclusively

    • 4 important sequences in leader sequence

      • Alternative base pairing between them results in different results

      • 1-2 (which leads to 3-4 binding), 3-4 lead to 3-4 terminators stem loop

      • 2-3 leads to antiterminator stem loop (prevents 3-4)

      • If 3-4 pair: structure forms → attenuator → acts as a transcription terminator

      • 2-3 pair → 3-4 cannot pair → attenuator not formed → 2-3 pair has no effect

      • Ribosome stalls at sequence 1 when trp is low → leads to 2-3 binding

      • leader region is completely translated in 3-4 binding, trp is high so passes through sequence 1 quickly leading to 3-4 binding

    • When leader region is completely translated → 2-3 reforms after passing through stem loop created by 2-3

Lecture 4:

• Prokaryotic DNA vs Eukaryotic DNA: 

  • Information density: prokaryotes greater than eukaryotes

    • More genes/bps in prokaryotes

  • Eukaryotes have repeating DNAs → prokaryotes have unique DNA

  • Genome association w/ protein (Chromatin)

    • Prokaryotic genomes loosely associated

    • Eukaryotic genomes tightly associated with histones and chromosomal proteins: chromatin

• 1% of human genome encodes for proteins

• As organism complexity increases the genes per base pair decreases

  • Eukaryotes are incredibly complex organisms with very large non-coding regions

• Renaturation kinetics/Reassociation kinetics

  • Help us understand why DNA is repetitive

  • Eukaryotes need to package data

    • Compacting DNA vs reliable access to DNA → chromatin is solution

      • Proteins include specialized structural proteins/enzymes

      • 30 nm chromatin fiber organized in loops that can be individually opened (looped domains)

  • Sequences will have plateaus depending on their repetitiveness in kinetics chart

    • Very repetitive sequences will anneal fastest

    • Very unique sequences will anneal slowest

      • Prokaryotes have 1 jump in transition

      • Eukaryotes may have multiple jumps

        • (3 classes/3 rises, 4 rises/4 classes, etc.)

• RNA polymerase in Eukaryotes and Prokaryotes

  • 3 eukaryotic RNA pol vs 1 prokaryotic RNA pol

    • Eukaryotic RNA pols: rRNA, mRNA, and tRNA synthesizers 

  • Eukaryotic RNA polymerase requires multiple transcription factors while prokaryotic require one or two at most

  • Eukaryotes have post-transcriptional processing that involves capping, splicing, and polyadenylating mRNA

    • Prokaryotic DNA generally does not contain introns

  • Eukaryotic RNA pol requires factors to modify chromatin/looped domains to access genes

    • Bacterial genes are not packaged like eukaryotic DNA so it can be easily accessed by RNA polymerase

• Eukaryotic RNA polymerases

  • Polymerase:

    • 1 → resides in the nucleolus (innermost portion of nucleus)  → transcribes pre-rRNA typically

    • 2 → Nucleoplasm → pre-mRNA and some snRNA

    • 3 → Nucleoplasm → tRNA, some rRNA, some snRNA(spliceosome), and signal recognition RNA 7SL RNA

• Eukaryotic core promoter motifs

  • TATA box

    • TATAXAX consensus sequence

    • 25-30 bp upstream of transcription start site

  • Initiator element (Inr)

    • Overlaps with transcription initiation site

  • DPE

    • Downstream promoter element

    • Extends from about +28 to +34

  • TFII recognition element (BRE)

  • Typically the order seen is the BRE → TATA Box → Inr → DPE

• General transcription factors (GTFs) form the preinitiation complex (PIC) 

  • DAB F Pol EH (District Attorney Beats Four Policemen Eating Hamburgers)

  • [TFII (prefix for each)] D→A→B→F-Pol(joins together)→E→H

    • ^^assembly order^^

  • TF = transcription factor

  • II = RNAP II 

  • TFIID

    • Made up of TBP(TATA-binding protein) and TAFs (TBP-associated factors)

    • Very large

    • First to bind

    • Binds DNA in the minor groove

  • TFIIA

    • Recognizes core promoter 

  • TFIIB

    • Recognizes core promoter

  • TFIIF-Pol

    • F targets the Polymerase to the promoter

  • TFIIE

    • Modulator of helicase

    • Binds after Pol/TFIIF binds to preinitiation complex

    • 2 different subunits/both needed to stimulate transcription

  • TFIIH

    • Helicase

    • 9 subunits

    • DNA helicase activity/ATPase activity

    • Kinase activity → phosphorylation of CTD of the large subunit of RNAP

    • CTD protein Kinase

• Xeroderma pigmentosum

  • Mutations in TFIIH subunits lead to extreme light sensitivity and ultimately cancer

  • May lead to death in childhood

  • Cockayne syndrome is the same mutation

• REVIEW OF EUKARYOTIC TRANSCRIPTION INITIATION

  • Low density of coding information

  • Large amounts of introns

  • 3 RNA polymerases

  • Pol I and III transcribe rRNA and small RNAs have unique promoter requirements

  • Pol II transcribes mRNA which leads to gene expression

  • RNA pol II promoters assemble GTFs (General transcription factors)

  • Preinitiation complex(PIC) consists of more than 30 individual proteins

    • PIC does initiate transcription at very low activity

• CTD (Carboxyl terminal domain associated with 3’ end of reading frame for mRNA) of large subunit of RNAP helps transition from initiation to elongation 

  • CTD contains many tandem repeats of the heptapeptide Tyr-Ser-Pro-Thr-Ser-Pro-Ser

    • Number of repeats ranges from 26 in yeast to 52 in humans

    • Repeats can be phosphorylated

    • Phosphorylation state differs at different stages of transcription

    • Ser 2 and Ser 5 are imporant

• Transcription Elongation

  • Largest subunits of RNA pol I, II, and III as well as E. Coli RNA polymerase have many homologies

  • Pol II is unique in having a CTD with heptapeptide repeats

  • CTD is indispensable → deletion mutants are lethal in yeast

  • RNA pol II CTDs have to be unphosphorylated for the PIC to form

  • TFIIH has to phosphorylate RNA pol II CTDs for RNA elongation to occur

  • Serine 2 and serine 5 are very important in CTD

    • RNA pol II is phosphorylated at ser5 to initiate elongation

    • RNA pol II is phosphorylated at ser2 after bp +50 during elongation

  • RNA pol II 

    • O = phosphorylated → elongation ensues

    • A = unphosphorylated → no elongation

    • When it is phosphorylated DAB is then kicked off

• 2 proteins regulate elongation as well

  • NELF → Negative elongation factor 

  • DSIF → DRB-Sensitivity-Inducing-Factor

    • DRB is an inhibitor of CDK9 which is a component of the positive transcription elongation factor (P-TEFb) 

  • ATP then is used to knock off NELF and DSIF

    • then elongation can be induced by the P-TEFb

• Transcription Elongation factors

  • Fork loop 1

    • prevents premature unwinding

  • rudder

    • prevents the DNA to rebind to mRNA

  • lid

    • wedge and guide the incoming DNA

  • bridge helix

    • acts as a ratchet (circular motion in one way)

  • more than 100 proteins associated with transcription of RNA polymerase II

Lecture 5:

• mRNA processing (3 steps)

  • 5’ Cap needs to be added

  • Splicing the mRNA

  • Cleavage/Addition of the Poly-A tail

• 5’ Cap (pay attention to enzymes below)

  • Function

    • Protects mRNA from nucleases

    • Distinguishes mRNA from other types of RNA

    • mRNA export

    • Translation initiation (Ribosome binds to the 5’ cap)

  • How its made

    • 5’-triphosphate Step 1

      • H2O → H2PO4

      • The 5’ phosphate is removed and now we have a diphosphate at the end

    • Guanylyltransferase Step 2

      • GTP → PPi

      • Guanosine is added

    • N7G-methyltransferase Step 3

      • S-adenosylmethionine → S-adenosylhomocysteine

      • Guanine is methylated

    • Capping enzyme is recruited by the CTD of RNA pol II

      • CTD must be phosphorylated on Ser-5 (negatively charged attracts positive enzyme)

      • CE = Capping enzyme

        • Bifunctional

        • RNA triphosphatase and guanylyltransferase

• mRNA Splicing

  • Function

    • Allows many proteins to be produced by 1 gene

    • mRNA export

    • Translation importance (nonsense mediated decay)

    • Introns are removed

  • Alternative Splicing

    • pre-mRNA can be spliced differently to produce many different mRNAs 

    • Errors may lead to muscular dystrophy

  • 4 elements for splicing

    • GU-rich sequence is the 5’ splice site

    • AG sequence 3’ splice site

    • Branching nucleotide

      • Adenine

      • Most important

    • Pyrimidine rich tract

  • Splicing steps

    • 2’-OH of adenine is the nucleophile

    • The Branching Nucleotide’s 2’-OH attacks the 5’ GU

    • 3’-OH of the 5’ GU that was removed attacks the AG at the 3’ splice site

    • Exon is separated from intron

  • Spliceosome

    • Promotes the splicing and is made up of snRNA

• Transcription Termination

  • Cleavage at poly(A) site

  • Addition of the poly(A) tail at 3’ end

  • Transcription termination downstream from cleavage site

  • Functions of poly(A) tail

    • Protects from exonuclease activity

    • Important for transport of mRNA

    • Important for translation

    • Allows for isolation of mRNA in a lab

• Antitermination model for termination vs Torpedo model of termination

  • Antitermination is less supported than torpedo model

  • (likely incorrect) Antitermination proposed that there was a antiterminator that attached to the RNAP II and prevented the release of the enzyme until hitting terminator factors that would elicit the release of the RNAP from the DNA

  • (likely correct) RNAP is stalling and slowing down after making polyA tail → RAT1/Xm2 attacks the poly(A) tail → ends the termination after degrading rest of mRNA (torpedo model) (enzymes involved are important)

• mRNA export requires GTP hydrolysis

  • mRNA out needs Exportin and Ran + 1 GTP

  • mRNA in needs importin and Ran + 1 GTP

• Eukaryotic Transcription Regulation

  • Sequence specific transcription factors

  • Promoter elements

    • Eukaryotic promoters are OFF in the absence of regulatory factors

    • Bacterial promoters have a basal/low transcription rate

  • Enhancers

  • Activating and repressing mechanisms

  • Mediators 

• Chromatin remodeling

  • Allows transcription → loops of DNA are unpacked in order for transcription to initiate

  • Histones: basic proteins that package and order eukaryotic DNA into units called nucleosomes

  • Euchromatin → loosely packed DNA (more expressed

  • Heterochromatin → tightly packed chromatin

  • Transcribing a gene → 

    • HATs encourage transcription by decreasing the affinity of nucleosomes → leading to less tightly bound DNA to histones

    • HDACs increase the affinity of histones for DNA → more tightly bound to each other

• Eukaryotic promoters must be activated → RNA polymerase have little to no affinity for  promoters without additional factors

  • Combinatorial control: specific combination of transcription factors must be bound at the promoter in order to express a specific gene → large eukaryotic promoters can bind many transcription factors 

  • General TFs bind at core promoter

  • Transcriptional activators that bind DNA and co-activators which bind the activators bind at regulatory sequences both upstream and downstream

• Cis vs Trans activiating elements (not to be confused with the chemical terminology of cis- and trans- conformations)

  • Cis-repsonsive elements are elements within the DNA sequence that are not the promoter but bind transcriptional factors

  • Transcription factors or (trans-acting regulators): are proteins that bind cis-responsive elements

• Transcription factors

  • bind to enhancers(DNA) and mediators(proteins)

  • Contribute to the chemical modification of the PIC

  • Stimulate elongation of RNAPII

  • Act on the level of chromatin

• DNA binding and activation/repression domains:

  • Transcription factors have a bimodal composition

  • One domain recognizes a specific DNA motif or “DNA binding domain”

    • Zinc-finger sequence motif

      • Binds to major groove

      • Complex formation between 4 cysteine/histidine residues and a zinc ion

    • Helix-turn-helix motif

      • Binds to major groove

      • Form dimers (if one of the proteins is mutated it will not function properly)

      • 2 helices → one recognizes and fits into the major groove 

    • Leucine Zipper

      • Leucines are spaced 7 proteins apart

      • Dimers as well

  • Second domain affects transcription activation

  • This can lead to many more combinations of Transcription factors such as if there are 8 transcription factors you can have 64 combinations

• Eukaryotic promoters:

  • Sigma factor helps bind RNAP to binding sites in prokaryotes

  • eukaryotic promoters are

    • Sequences bound by PIC (Core promoter)

    • binding sites for transcriptional activators (regulatory promoters)

      • Interacts with a specific target sequence which is sometimes close to the transcription start site

• Transcription is controlled by a promoter and an enhancer

  • Separation from enhancer may be in several kbps to the promoter VERY FAR

  • Enhancer

    • Location and orientation of enhancer is independent and functions at a distance

    • Enhancers can be upstream or downstream and still provide the same function

  • DNA looping occurs between regions bound to activator proteins

  • DNA binding domain on an enhancer is bound by an activator which then binds to the PIC which is bound to the core promoters (TATA,Inr,DPE)

• Enhancers and Silencers

  • Activators(protein) bind to enhancer sequences

    • Determines which genes are switched on and increase the speed of transcription

  • Repressors(protein) bind to silencer sequences

    • Interferes with the functioning of activators and slows transcription

    • May either interfere with the binding site of the activator (the enhancer sequence) (competitive DNA binding)

    • may bind to the activator as it may “mask the activation surface” of the PIC

    • May bind to the GTFs in the PIC and “directly interfere with the binding of the activator” on an enhancer sequence to the PIC

  • Enhancers(cis element) need a mediator(trans element)

    • Specifically interacts with the CTD of the large subunit of RNA pol II

    • CTD attracts the attachment of many additional factors including

      • Termination factors, splicing factors, elongation factors, mediators

      • CTD acts as an assembly line for tools needed for promoter clearance and RNA processing which is coupled to the elongation process

      • mRNA might get fed through this line of factors which are bound to the CTD

    • Binding of an activator to an enhancer recruits RNAP II through mediator/RNAP complex

    • When many enhancers aim to promote transcription their effect is synergistic

      • I.e. 1 activator may produce a synergistic effect of 1 unit

        • 4 activators may produce a synergistic effect of 500 units

  • Insulators block activation by enhancers, or block repression by silencers.

Lecture 6:

• Chromatin packaging hierarchy

  • Nucleosome forms (DNA + Histones)

  • 30 nm fiber (coiled nucleosomes)

  • Nuclear scaffolding w/ looped domains

  • Metaphase chromosome (mitotic)

• Histones made up of octamer (2xH2A,H2B,H3,H4)

  • 146 bps wrap around a histone (2 turns)

  • Histones contain many basic amino acids (Lysine & Arginine)

    • Histones as a result are positively charged

    • DNA is negatively charged

    • Strong electrostatic interactions

• DNA packaging + transcription

  • Heterochromatin vs Euchromatin 

    • Euchromatin = histones on the outside of the plane which allows for transcription

    • Heterochromatin = histones are tightly wrapped and adjacent limiting transcription

• How to alter DNA packaging

  • Chromatin Remodeling

    • Chromatin remodeling complex exposes promoter that may be bound to nucleosome

    • Brings out the promoter to the string

  • Histone tail modifications

    • Histone tails can be modified to create different changes to the histone itself

      • Histone modifications influence transcriptional activity

      • Acetylation

        • HATs (histone acetyltransferase)

        • Lys residues at N-terminus

        • Enhances transcription by destabilizing nucleosomes

      • Deacetylation

        • HDACs (histone deacetylases)

        • Stabilizes compact chromatin structures

        • Represses transcription

      • Methylation 

        • Lys and Arg residues

        • Repression

      • Phosphorylation

        • Ser residues

        • Activation 

      • Ubiquitination

        • Lys residues

        • Activation

  • Remodeling and Modification usually work together

  • Chromatin Remodeling Complex is SWI/SNF

• Methylation

  • Chromodomain

    • Allows proteins to bind methylated histones

  • Chromoshadow domain

    • Allows HP1 to bind to other HP1 Protiens

• Epigenetic code 

  • Covalent changes to histones and DNA create changes that may alter gene expression and result in being read like the genetic code

• Transcription Regulation and Cancer

  • Cofactors

    • Binds transcription factors without making DNA contact

    • Can be super-activators (cofactor)

    • Can repress the activity of the TFs (corepressor)

      • E2F → constitiutive transcription factor in the context of certain cellular genes

      • Binds the cofactor RB and loses activation function

  • Cell cycle is regulated by transcription factors and co-repressors

    • S phase = replication of chromosomal DNA 

    • G1 phase = no replication of chromosomal DNA

    • G1-S transition: express proteins needed for replication of DNA like DNA polymerase

      • G1 co-repressor(RB) blocks E2F function

      • Regulated phosphorylation of RB leads to it’s removal and reactivation of E2F → leading to S phase

    • In many cancers the RB gene is mutated and cells permanently go from G1 to S without stalling

      • RB mutations may lead to cancer

        • RB originally found mutated in cancers of the retina

        • Some of the most frequent mutations leading to cancer

      • Transcription regulation is central to carcinogenesis

        • Normal cells have cdk2 to phosphorylate RB and remove it from E2F to have typical DNA replication

        • Cdk2 is negatively controlled by a different factor (p21CIP)

      • p21 transcription is activated by p53

      • p53 availability determines E2F repression by RB

      • p53 is a tumor suppressor → loss of function/mutation would potentially lead to uncontrolled tumor proliferation

        • p53 available(up) → p21 up → cdk2 down → RB up → E2F down → G1 does not go to S phase (inverse is true for p53 mutation)

      • Many viruses target p53 and RB and cause cancer (papillomaviruses, polyomaviruses, and adenoviruses)

• siRNA

  • mRNA + tRNA and → 

    • small stretches of RNA which complements part of mRNA (siRNA)

    • Double stranded RNA is responsible for lowering RNA levels

  • RNAi → RNA interference

  • Long ds-RNA (double stranded)

    • RISC(big complex that contains dicer and argonaute) → Dicer cleaves ds-RNA and siRNA

      • Protein breaks strands into single strands

        • Delivers to RNA

          • Transcript is degraded

  • Small RNAs / RNA interference

    • Previously seen that small RNAs exist in the cell

    • Problem → small RNAs found in the cell siRNA or degradation products of other larger RNAs 

      • Are they natural?

        • Yes → are they degradation products or siRNA tho

          • Northern Blot → sequence of small RNA

            • Sequence found in genomes are very similar sizes

              • NOT random degradation

    • Endogenous RNA → microRNAs(miRNA)

      • 21-23 nucleotides

      • Primary mRNA transcribed by RNA pol II

    • Different parts of RNA interference

      • RISC → RNA interference is done by this complex

      • Dicer → cleaves the DS RNA

      • Argonaute → cleaves between siRNA and mRNA

    • Drosha processes microRNA

      • pre-miRNA then is loaded onto RISC

        • Processed by RISC

    • siRNA vs miRNA 

      • siRNA is artificial (Gene silencing)

      • miRNA is encoded by genome (RNAi)

      • miRNA leads to translation inhibition

      • siRNA leads to translation inhibition and/or degradation

• CRISPR-Cas

  • Clustered Regularly Interspaced Short Palindromic Repeats

    • Used by bacteria to fight viruses

  • 3 steps

    • Acquisition - cas locus → binds to virus DNA → GGG sequence → cuts 20 bases upstream

    • Expression - bait the DNA from the virus 

    • Interference - Cas protein bind to CRISPR DNA 

  • Similar to siRNA but the difference is it involves DNA

  • CRISPR and Eukaryotic cells → possible to create specific genome modifications

    • Required are Cas9(nuclease), Gene specific CRISPR RNA (crRNA), and tracr RNA → links crRNA to Cas9

• 3 Genome editing techniques 

  • Zinc finger (motif)

    • Binds DNA and cuts with endonuclease (FolkI) 

    • Issue is that zinc finger has to be remade each time

  • TALEN (helix-turn-helix)

    • Binds DNA and cuts with endonuclease (FokI)

    • Issue is 9 proteins needed to be bound

  • Cas9/CRISPR

    • gRNA binds DNA, cuts by an externally added endonuclease (Cas9)

    • Allows highly specific binding and linked to trcrRNA

    • BEST strategy 

    • gRNA??? → guide RNA → crispr RNA

    • RNAi inhibits an RNA, CRISPR/Cas9 will inhibit DNA (major difference)

• Human collections generated by Cas9/CRISPR include genes essential for cancer cells and genes important for resistance to chemotherapy drugs

Lecture 7:

• Translation by ribosome

• Proteins differ from nucleic acids:

  • 20 amino acids vs 4 nucleic acids

  • Large variety of functional groups

  • Accelerate a multitude of chemical reactions

  • well-defined tertiary structure (shapes)

• 4 nucleic acids ^ 3 codon slots → 64 combinations of nucleic acids

• tRNA

  • Transfer RNA

• Ribosome

  • Factory to make

• How is code read?

  • Unpunctuated code → deletions of 3 nucleotides would restore the reading frame

  • The two wrong proposals were overlapping code and punctuated code

• Deletions

  • 1-2 frameshift mutation

  • 3 nucleotides = removal of 1 aa

  • Insertion of 3 nts → insertion of 1 aa

  • Change of 1 nt results in a missense or nonsense mutation

• Filter experiment

  • Synthetic mRNA that codes for certain amino acids added to solution

• Filter binding assay

  • Filter contains mRNA and only the corresponding tRNA would attach to filter → aa that is radioactive would then be stuck on filter

• There are variants to the genetic code

  • UGA = Trp, AUA = Met, AGA = Stop in mitochondria

• Genetic code

  • Non-overlapping, no spacers

  • Almost universal

  • Highly degenerate = many aas are specified by two or more codons

  • Unambiguous = codons specify ONE aa

• Crick’s adaptor hypothesis

  • tRNA

  • AA – Adaptor(tRNA) – Nucleic acitd

• tRNA cloverleaf secondary structure

  • Amino-acid arm - conserved CCA_OH → attaches to AA

  • Anticodon arm - read antiparallel to mRNA

  • D-arm

  • Extra arm

  • TψC arm 

• 7-15% of tRNAs contain modified nucleosides

  • Post Transcriptionally modified

  • Adenosine at 5’ anticodon changes to I (Inosine) → can base pair with A, U, C → “Wobble theory” / third base wobble

    • tRNA ala found to bind to GCA, GCC, and GCU → Inosine is the 3rd base

    • Inosine is highly ambiguous

    • Allows for more conservation of energy

    • 32 tRNAs for 61 sense codons (3 are stop)

    • Non-canonical base pairing in one base provides weaker interaction between anticodon and codon → higher rate of protein synthesis

• tRNA folds into a L shaped 3D structure

  • Active sites include anticodon and amino acids and they are maximally seperated

  • The anticodon stem and acceptor stem form double helices

• Aminoacyl-tRNA synthetase

  • Matches tRNA vs AA

  • AA attacks ATP → becomes adenylated

    • Aminoacyl-AMP formed → 2’ OH of tRNA attacks C which releases AMP (which is a great leaving group)

• How do aminoacyl-tRNA synthetases know they have the correct AA attached?

  • AA too large → does not fit

  • AA too small → fits → proofreading site within enzyme → removed through hydrolysis

Lecture 8

• Ribosome History

  • Who won the ribosome race?

    • Tom Steitz

  • Who first crystallized the ribosome?

    • Ada Yonath

• Ribosome

  • 4 binding sites

  • Large subunit

    • E - Exit

    • P - Peptide

    • A - Amino

  • Small subunit

    • mRNA binding site

  • 2/3 of the ribosome is RNA and 1/3 is protein

    • Proteins are just there to stabilize the RNA

• Translation

  • aa + ATP + tRNA → aa-tRNA + AMP (hydrolysis of ATP = loaded aminoacyl tRNA)

  • Bacterial initiation utilizes fMet (formyl-Methionine) and initiator tRNA

    • fMet is loaded by 2 enzymes → Methionyl-tRNA synethetase & Methionyl-tRNA formyltransferase

    • Difference between fMet and Met tRNA is that there is a mismatch between AC which provides a kink !!!DIFFERENCE IS IMPORTANT!!!

      • fMet-tRNA to AUG (loads to P site) → regular methionine would load to A site

  • fMet-tRNA binds to AUG

  • Prokaryotic translation

    • 3 initiation factors and GTP hydrolysis

    • 30S subunit (small subunit)

      • IF-1 Bound to the amino site

      • IF-3 bound to nowhere (in relation to APE) (E site bound during initiation)

      • Order bound is:

        • IF-1 → IF-3 → mRNA → then IF-2 binds (explained below)

      • Shine-Dalgarno (SD u can write SD so it’s prolly gonna be on the exam) sequence complementary to the 16S rRNA

        • 16S rRNA is within the 30S subunit (small)

          • Made up of other proteins as well

      • fMet-tRNA is accompanied by the IF-2 that binds to IF-1 which is within the A site

    • So what happens in order now is after the IF-1 binds to the A site and IF-3 binds to the E site → mRNA is now fed through the ribosome in which the SD sequence complements to the 16S rRNA → and the IF-1 is now within the A site → IF-2 binds to the IF-1 within the A site and carries the fMet-tRNA along with it.

      • After all this happens the 50S subunit binds to the 30S subunit making the 70S ribosome and knocks the IFs off the small subunit

        • Something to note here is that fMet still remains on the tRNA that now resides within the P site

    • To clarify once more:

      • IF-3 is nowhere until initiation

      • IF-1 is in the A site

      • IF-2 is on top of IF-1 (bc it binds later)

      • fMet-tRNA is in the P site (comes in with IF-2)

  • Riboswitch inhibition → feedback inhibition

    • If an end product M is present → loop formed at AUG → ribosome falls off → translation is OFF

• Eukaryotic Translation Initiation

  • Many eIFs → no fMet

  • Eukaryotic initiator Met-tRNA has unique features

    • 12 different IFs

• Eukaryotic translation steps

  • Small subunit complexes with eIF1, eIFA, eIF3

  • Initiator Met-tRNA w/ eIF2 and Met binds to complex w/ eIF5B-GTP

  • mRNA 5’ cap binds to eIF4F 

  • mRNA-eIF4F binds to the preinitiation complex

    • The Kozak Sequence establishes the reading frame → binds to eIF4F

    • polysome formation involves 5’ cap and poly(A) tail

    • rRNA binds to mRNA through base pairing

    • Scans till AUG

  • Translation Initation complete

  • Locations of each of the eIFs is more important though

    • eIF3 is bound to nowhere (on the opposite side of the ribosome of the APE site)

    • eIF1 is bound to E site

    • eIF2 is bound EXCLUSIVELY to the Met-tRNA (does not touch the ribosome)

    • eIF1A is bound to the A site

    • eIF5B is bound to eIF1A which is bound to the A site

    • Met-tRNA is bound to the P site

    • eIF4F binds to the mRNA’s 5’ cap → which then binds to the ribosomal subunit (does not bind to a site)

• eIF2 mutation leads to huntington’s disease

• Elongation

  • Elongation factors are needed for chain elongation

  • Steps

    • 1. aminoacyl-tRNA binds to A site

    • 2. tRNA moves over to the P site and a new peptide bond is formed

    • 3. ejection of spent tRNA from the E site

  • Ribosome catalytic base is an Adenine

  • prokaryotes:

    • fMet-tRNA binds to the P site and and GTP-Tu binds with the second aminoacyl-tRNA at the A site → GTP hydrolysis → Tu falls of and forms Ts-Tu complex → GTP then binds to another Tu to continue the process

    • Peptide bond is formed → aa chain is at the A site 

    • EF-G-GTP binds the A site

    • Translation requires GTP hydrolysis

  • Puromycin binds to A-site

• Transcription if from 5’ to 3’, ribosomes move 5’ → 3’

• Termination

  • Protein is made → Stop codon

  • A site scans for stop codon → binds RF2 (release factor)

    • binds to A SITE

    • GTP hydrolysis is involved in termination

      • hydrolyzes polypeptide

  • RFs 

    • RF1 recognizes UAG or UAA stop codon

    • RF2 regonzises UGA or UAA stop codon

    • RF3 - stimulated the rate of peptide release by RF1/RF2 but does not act independently

    • difference is not tested

  • eRFI (human translation release factor)

    • looks like a tRNA w/ and L shape

    • eRFI, RF1, and RF2 all bind to the A site

  • tmRNA

    • compare it to tRNA^Ala

  • ribosome rescue pathway (method 1)

    • tmRNA binds to A site

    • Ala on tmRNA attacks the polypeptide chain

    • frees the ribosome from the polypeptide chain

  • Ribosome Recycling (method 2)

    • RRF binds !!!A site!!! (just pay attention to A site bascially w/ termination)

    • EF-G-GDP binds to A site

      • pushes RRF to P site

    • Then IF-3 comes in to the E site and kicks RRF and EF-G off