Molbio EXAM 2 (kill me kill me kill me)

AHHHHHH WHAT WHY SO SOON WE JUST HAD ONE

How are nucleosomes replicated?

All nucleosomes come off and reassemble on new DNA

All old nucleosomes get distributed on both daughter strands (then new histones are added to form new nucleosomes)

How do old nucleosomes “move”

FACT (Facilitates Chromatin Transcription) is a histone chaperone.

—> binds to H3:H4 tetramer, transfers it behind the replisome

FACT is assisted by ASF1 (anti-silencing factor)

—> interacts with helicase at fork

New nucleosome modification?

ACETYLATION of N-terminus!! To make new DNA more accessible

Regulation of DNA replication

First: activation of origin when needed

Second: preventing the origin from being used until after cell division

All cells need to be regulated by the readiness of the cell for replication and division

Origin firing in bacteria (What does it mean?)

Becoming active (all parts activate)

What happens in origin firing in bacteria? FIRST WAY (DNA A and ATP)

DNA A hydrolyzes ATP when a clamp-associated protein interacts with it

*DNA A is bound tightly to ADP and does not bind DNA tightly

→ Either new DNA A must be synthesized, OR ADP must be removed

What happens in origin firing in bacteria? SECOND WAY (Seq A)

Seq A Blocking

1) DnaA binds fully methylated DnaA boxes on GATC

2) After initiation and ATP hydrolysis by DnaA, DnaA boxes are hemimethylated

3) Seq A binds to hemimethylated DNA at the origins and prevents DnaA binding

4) Bound Seq A will dissociate from DNA, and a new SeqA can bind or Dam methylate can bind and methylate DNA

5) Fully methylated chromosomes segregate to daughter cells and are now competent for DnaA binding and a new round of replication

What happens in origin firing in bacteria? THIRD WAY (DAT)

DAT

DAT binds dnaA more tightly than oriC

→ After replication, there are 2 DAT sites

→ After cell division, there is only 1 site

After cell division, DnaA:ATP levels rise

→ Newly synthesized DnaA

→ Interaction of DnaA with DARS

• DnaA Reactivating Sequence

Eukaryotic regulation at origins

“Licensing” followed by “Firing”

Origin firing based on DNA:ATP, 3 regulations

1) Amount of Dna A:ATP has to increase

2) Methylation of GATC sequence at origin required

3) Delay of methylation through blocking of the methylase by Seq A

Eukaryotic Licensing

D6 associates with part of helicase and CDT1

mcm2-7 is recruited by ORC/CDC6, protease degrades cdc6 & cdc1 → more mcm’s recruited

*bidirectionally oriented mcm2-7 helicase pair

!! More origins are licensed than are used

Eukaryotic Firing

cdc45 & DLD3 are activated by S-phase DDK

pol ε, SLD2, GINS (五一二三) activated by S-phase CDK

→ Requires phosphorylation by DDK (regulated by CDK)

CREATES: a helicase complex called CMG helicase

DNA pol vs RNA pol (compare and contrast)

DNA: Dumb

RNA: Occurs throughout cell cycle, A-U & G-C, Txn ‘bubble’ (small unwound region in enzyme)

Same: Add to 3’, Break double helix (H-Bonds), Base pairing

Steps in transcription

Promoter recognition

→ Forms the “closed complex”

Promoter opening

→To form the “open complex”

Initiation (first bonds of RNA made)

Promoter clearance

→ Loses contact with the promoter

Elongation

→ Must be very processive (because incorrect base pairs can change DNA or protein)

Termination

RNA Polymerases

• Bacteria and archaea have a single RNA pol (called RNAP)

• Eukaryotes have at least three (pol I, pol II and pol III)

Remarkable similarities in all pols

• All contain a core enzyme that can’t recognize promoters but makes the RNA

• All contain multiple subunits

• All have accessory protein(s) that help bind to promoter

2 FORMS:

‘core’

‘holoenzyme’ (binds to promoter)

Task division in eukaryotic polymerase

• Pol I

→Transcribes only ribosomal RNA

• Pol II

→ Transcribes all protein-encoding RNA and most regulatory RNAs

• Pol III

→ Transcribes small RNAs, including tRNAs and some regulatory RNAs

How to isolate RNA polymerase to study?

• Break open cell (bacteria) or isolate nucleus (eukaryote)

→ Use protein purification

• Use assay

→ Look for things binding to promoters, production of RNA containing bacteria

Bacteria, Archaea, Eukaryote RNA pol have similar structures and jobs

• Common subunits

• For eukaryotic RNA polymerase, some common subunits, some unique ones

• Evolutionary: RNA pol has become more complex (more subunits)

• Pol II & archaean subunits have same #

How to determine the DNA sequence that makes DNA a “promoter”

→ Use chip seq

→ Make mutations

→ Compare sequences and look for consensus (valuable in bacteria)

2 main promoter sequences in bacteria

• -10 from promoter TATAAT

• -35 from promoter TTGACA

(These are the most common, 16-18 base pairs of non-consensus sequence in between)

! Proteins don’t usually match consensus exactly due to different proteins having different functions/ structures

UP Element

• UP element (upstream promoter element) increases txn

→ AT rich region beyond -35

→ Only found in the strongest promoters

What is required to recognize bacterial promoters?

SIGMA!!

→ Several sigmas named for molecular weight

→ normal sigma: σ70

Others:

• σ32 (heat shock)

• σ54 (nitrogen starvation)

• σ28 (flagellar genes)

Different promoter = different sigma factor (same job)

!! ALWAYS ASSOCIATED WITH RNA POL

The core RNA pol associates with a sigma factor that binds to a specific promoter sequence in the DNA, contacting sequences centered at the -10 and -35 regions.

The α C-terminal domains contact the UP element (located upstream up the -35 region of some promoters)

Purifying the Eukaryotic σ

RNA pol II could not start at promoters. HYPOTHESIS: there must be a “sigma factor”

→ Make nuclear extract, assay for correct txn if added pol II

→ Separate nuclear extract into fractions

→ Look for consensus sequence

FIRST THING FOUND: TATAAA 25bp before start of RNA (interacting with sequence, place RNA pol where RNA starts)

Promoter sequence besides TATA? (How they found σ?)

If you replace TATA sequence, transcription could still be active.

Through mutagenesis and sequence analysis, people found the INITIATOR

RNA Pol II could not start @ promoters

→ Make nuclear extract, assay for correct txn if added pol II

→ Find different subunits. What do they do?

DONT FORGET INITIATOR AND DPE (downstream promoter element)

TF2D binds to promoter

TF2B binds to B-recognition element & TF2D

TF2F interacts with TF2B & pol II

TF2E & H join

H & F unwind DNA with pol II

Eukaryotic transcription factor

TFIID

• Made up of TBP (TATA Binding Protein) and TAFs (TFIID Associated factors)

• Responsible for recognizing most parts of the core promoter

• TAFs are also responsible for recognizing most parts of the promoter

Multiple polymerases in eukaryotes (what are their transcription factors called?)

• Pol II

  • TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH

• Pol III

  • TFIIA, TFIIB, TFIIC

• Pol I

  • SL1 and UBF (does not follow naming pattern)

Pol I and III promoter elements

• Each has separate promoter elements

• Pol I (rRNA only)

→ UCE and core

• Pol III (tRNA and 5S rRNA)

→ Box A and Box C

→ TBP, TF3A, TF3C

! BOTH pol I and pol III required TBP (TBP bends DNA at promoters)

even though theres no TATA to bind to

Promoter Clearance

RNA polymerase changes conformation after making a short chain

Bacterial structural studies (elongation and sigma)

• Sigma interaction with the core disrupted as RNA extends

• Gradually loses interactions

• Eventually lost after elongation progresses

• Sigma is not required for elongation

Eukaryotic promoter clearance (elongation and TFs)

• TFIIH phosphorylates CTD of pol II

→ CTD = carboxy terminal domain (Contains repeats of Tyr1–Ser2–Pro3–Thr4– Ser5–Pro6–Ser7)

→ TFIIH phosphorylates Ser5

• This allows factors to bind to allow for 5’ capping of RNA

• pTEFb phosphorylates Ser2 on CTD

• Elongation goes

Transcription bubble

• Topoisomerase keeps (+) and (-) going their respective ways

• RNAP: DNA:RNA “ternary complex” very stable

• Allows for highly processive elongation

• Other factors help!! (Elongation factors)

Elongation factors

• Many different functions

• Without proper function = cancer (elongate uncontrollably)

RNA pol backtracking

• Proofreading

• Can “restart“ if paused

• Requires factors

  • GreB (E. coli) → promotes transcription elongation by stimulating cleavage activity of the RNA polymerase.

  • TFIIS (Euks)

Termination

• Elongation must become unstable somehow

  • RNA removed from RNA:DNA hybrid

  • Transcription bubble collapse

  • RNA pol dissociation from DNA

! Different in bacteria and Eukaryotes

Termination in BACTERIA (2 ways)

1) Sequence Dependent

• Transcribed region contains self-complimentary sequence that can form a hairpin

• Then, a string of A’s in the template strand causes U’s in RNA

• Hairpin inserts into RNA exit channel, pauses RNAP

• RNA:DNA hybrid comes apart

2) Factor Dependent

• Requires a protein called “rho” (5’ - 3’ RNA helicase)

• Transcribed region contains a C-rich region to which rho binds

• RNAP pauses, rho catches up and RNA:DNA hybrid comes apart (rho pulls RNA out)

Eukaryotic termination (do not learn in detail yet)

! Differs for pol I, pol II, pol III

• pol I and pol III similar to bacterial sequence dependent

• pol II termination does not produce the 3’ end of the RNA

• Termination is linked to RNA processing

• Polyadenylation at the 3’ end actually determines the end of the RNA