Quiz 4 Content

Main differences between DNA and RNA

• 2’ -OH

• Uracil instead of Thymine

• RNA can fold up to look like a helix

• single stranded vs. double stranded

monocistronic

• codes for only 1 polypeptide

• in eukaryotes and bacteria

polycistronic

• codes for 2+ different polypeptides

• in bacteria and archaea

transcriptome

the sum of all the RNA molecules produced in a cell under a given set of conditions

mRNA

• encodes the amino aicd sequences of polypeptides

tRNA

read the mRNA and transfer the appropriate AA to a growing polypeptide chain during protein synthesis

rRNA

constituents of ribosomes, the cellular machines that synthesize proteins

ncRNAs

have a variety of catalytic structural, and regulatory functions

RNA polymerase

• catalyzes transcription

• requires a DNA template

• add ribonucleotide units to the 3’ -OH end

• has 2 Mg2+ groups just like DNA polymerase where a pyrophosphate leaves

overall RNA polymerase rxn

(NMP)n + NTP → (NMP)n+1 + PPi

template strand

DNA strand that serves as template or RNA synthesis

Nontemplate strand

• aka coding strand

• DNA strand that is identical in base sequence to the transcribed RNA, with U in RNA in place of T in DNA

promoter sequence

• directs RNA polymerase to a specific chromosomal location

• -35 region → - 10 region (TATAAT)

sigma factor

• recognizes the promoter sequence

sigma70 RNA polymerase

• binds between -70 — +30

RNA polymerase + supercoils

• a transcription bubble forms when the DNA duplex unwinds

• RNA polymerase generates positive supercoils ahead and negative supercoils behind

  • DNA can twist but cannot swivel, besides when topoisomerase is used

• RNA can exite through an RNA exit

Initiation + Elongation by E. coli RNA Polymerase (6 steps)

1. RNA polymerase binds to the promoter, directed by a sigma factor

2. Closed complex (bound DNA remails double-stranded) forms, followed by an open complex (bound DNA is partially unwound near the -10 seq.)

3. Initiation causes RNA polymerase conformational change that moves it towards the gene

4. promoter clearance follows by elongation

5. NusA protein competes with the sigma subunit to bind the polymerase. Leads to sigma 70 dissociating

6. Transcription is terminated

   1. NusA dissociates from the enzyme

   2. RNA polymerase dissociates from the DNA

   3. RNA polymerase is recycled

σ70 RpoD

housekeeping sigma factor

σ32 aka RpoH

stress-induced promotor from heat shock

• has different -10 — -35 region

σ54 RpoN

specific for the promoters that control nitrgen assimilation

Importance of RNA polymerase orientation

• orientation is dictated by the promoter sequence

• anti-sense strand = template strand or nontranscribed strand

• sense strand = transcribed strand

three main things that control protein activity

1. modification/ligand binding

2. subcellular localization

3. synthesis and degradation

Positive induction

molecular signal causes binding of activator to DNA, inducing transcription

positive repression

molecular signal causes dissociation of activator from DNA, inhibiting transcription

negative induction

Molecular signal causes dissociation of repressor from DNA, inducing transcription

negative repression

molecular signal causes binding of repressor to DNA, inhibiting transcription

Functional groups - major groove binding specificity

• make a code protein that can recognize w/ specificity

  • A: H-bond acceptor

  • D: H-bond donor

  • H: Other H

  • M: Methyl group

Functional groups - minor groove binding specificity

• 3 in the minor groove vs. the 4 in the major

Lac operon in E. coli

Lactose is used as an energy source. Uses beta-galactosidase to break it down (encoded by the lac operon)

negative regulation by Lac repressor

a. glucose high, cAMP low, lactose absent

b. glycose low, cAMP high, lactose absent

positive regulation by CPR

a. glucose high, cAMP low, lactose present

b. Glucose low, cAMP high, lactose present

Cofactors role in regulation in Eukaryotes

• interact with the transcription factor to remove the nucleosome

• recruits GTFs

Eukaryotic Homeodomain

• eukaryotic transcriptional regulators

• play a special role during development

Zinc Finger

• Zn2+ is coordinated to 4 Cys and/or His

• feels the major groove out to help with binding

• on its own it is weak, but many zinc fingers are substantially enhancing binding

Leucine Zippers

• an amphipathic alpha helix with a series of hydrophobic amino acid residues on 1 side and a hydrophobic surface between 2 polypeptides of a dimer

• Leu @ every 7th position

• about 1 every 2 turns (3.3 residues per rotation)

RNA polymerase I (eukaryotes)

• rRNA

• consitiuents of ribosomes, the cellular machines that synthesize proteins

RNA polymerase II (eukaryotes)

• mRNA

  • encode the amino aicd sequence of polypeptides

• ncRNA

  • variety of catalytic structures and regulatory functions

RNA polymerase III (eukaryotes)

• tRNA

  • reads the mRNA and transfer the appropriate AA to a growing polypeptide chain during protein synthesis

• 5s rRNA

• ncRNA

Chloroplast + Mitochondria’s DNA polymerase

• have their own RNA polymerase

• evidence to support the endosymbiotic theory

• all RNA polymerases have a common ancestor!

TATA binding protein (TBP)

• recognize eukaryotic promoters

• the first component to bind to the preinitiation complex (PIC) at the TATA box of a typical Pol II promoter

• used in Pol I II and III as well as archaea!

• similar to the role of sigma factors in bacteria

TFIID Role

• positions the TBP and Pol II on the promoter

• binds the promoter DNA where the TBP subunits are anchored in the DNA minor groove

TATA box promoter element

• typically located at -30

• TATA(A/T)A(A/T)(A/G) sequence

• recognized by the TATA binding protein (TBP)

• not very common

Assembly of eukaryotic transcription RNA polymerase II initiation complexes (w/ general transcription factors!)

• TFIID recognizes the promoter sequence (with help form TFIIA)

• TFIIB, TFIIE, TFIIF, and TFIIH bring Pol II to the promoter, and initiate DNA melting

• TFIIH uses ATP energy to ram the DNA into polymerase which melts the site of trancription initiation

• TFIIH also phosphorylates RNA polymerase

CTD - carboxy-terminal domain

• aka repeat central

• The largest subunit of RNA polymerase II has a repeating AA sequence at the carboxy-terminal end

• for human RNA polymerase II, a sequence of 7 AA is repeated 52 times

• thought to help tether Pol II to the general transcription factors

TFIIH

• helicase-like activity

• starts initiation by phosphorylating the CTD of RNA polymerase II, promoting melting

• because it phosphorylates, it is called a kinase

• TFIIH is also a kinase

Phosphorylation of the CTD in RNA Pol II

• the phosphorylation state changes during trancription

• when Pol II dissociates, the gene is dephosphorylated

Eukaryotic Transcriptional Activates and Enhancers

• can be very far upstream

• bind to the cofactors to help enhance gene expression

Pol II transcription cycle

• Pre-Initiation Complex (PIC): TFIIA, TFIIB, TFIID (TBP), TFIIE, TFIIF, TFIIH, and Pol II

• the CTD - carboxyl-terminal domain of Pol II consists of 26 yeasts to 52 human repeats of -YSPTSPS- AAs

• TFIIH phosphorylates the CTD to trigger Pol II to initiate transcription

1. Pol II is recruited to the DNA by the GTP, TFIID finds the promoter site and TFIIH directly binds it.

2. transcription bubble forms (w/ TFIIH which phosphorylates the CTD to melt it further")

3. CTD is further phosphorylated during initiation

4. Transcription elongation is aided by elongation factors after TFIIE and TFIIH dissociate

5. Elongation factors dissociate. The CTD is dephosphorylated as transcription terminates a process facilitated by termination factors.

Compare + Contrast promoters across the 3 domains of life

bacteria:

• -35 to TAAT box at -10

• Sigma factors are very common

• DNA lacks nucleosomes

Eukaryotes:

• TATA box at -30 while initiation begins at +1

• TATA boxes are uncommon

Archaea:

• Have regulatory factors that are Bacterial-like

• have initiation factors and an RNA polymerase that is Eukaryotic-like

Nucleosomes

• fundamental organizational units of chromatin

• contain 8 core histone molecules: 2 of H2A, H2B, H3, H4

• 150 bp are bound to nucleosomes

• left-handed coil

Histones

• Small, Basic Proteins

• make up a nucleosome

• H2A, H2B, and H3 are in an evolutionarily related gene family

• H1 binds to linkers and is repressive

  • binds to the nucleosome exterior near the linkers

H2A

• includes H2A.Z, H2A.X, macroH2A and more

• in eukaryotes

• H2A.Z is in the first nucleosome of a Pol II transcription unit

• phosphorylated H2A.X exists primarily at sites of DNA breaks (help in DNA repair)

• macroH2A is involved in transcriptional repression

H2B

• also includes H2B.1

• is in eukaryotes

H3

• also includes H3.2, and H3.3

• in both archaea and eukaryotes

• H3.3 is enriched near transcriptionally active promoters

H4

• no other family members

• in archaea and eukaryotes

Histone Amino-Terminal Tails (Epigenetics!)

• intrinsically disordered

• where most of the histone modification occurs

• amino-terminal tails likely interact with one another

• four main types:

  • acetylation (ac)

  • methylation (me)

  • ubiquitylation (ub)

  • phosphorylation (ph)

acetylation (ac)

• K

• H3K9ac, H3K27ac, H4K18ac

• function is opening chromatin for transcription

methylation (me)

• K and R

• H3K4me1: enhancer mark

• H3K4me3: first few nucleosomes of a transcribed gene

• H3K36me3: marks nucleosomes in genes downstream of H3K4me3

• H3K9me3: repressive mark of heterochromatin

• H3K27me3: dynamic repressive mark

Ubiquitylation (ub)

• K

• H2BK123ub: helps return nucleosomes to gene bodies during transcription

• H2AK119ub: repressive mark

Phosphorylation (ph)

• S and T

• H3S10ph: is involved in chromatin condensation

Chromosome territories

• subnuclear region that constrains the entire structure of each chromosome

  • little or no intermingling of DNA in different territories

Long noncoding RNAs (lncRNAs)

• play a functional role in defining the chromosome structure

  • many provide a scaffold for proteins

Condensin and cohesion

• holds chromosomal regions together

Active + inactive compartments

• Active (“A”) - compartments withh reduced chromatin condensation (active)

• Inactive (“B”) - compartments (heterochromatin) are highly condensed

  • deactivating and repressive

  • H3K27 methylation could occur here

  • H3K9 methylation for permanent storage

Topologically associated domains (TADs)

• large segments of DNA are organized in loops

• binding of CTCF, cohension, and topoisomerase II to bordering sites brings the DNA into these loops

• The TADs are relatively close

Epigenome

• regulates information flow

• backregulation

• includes nucleosomes with their modifications

  • Acetylation, methylation, phosphorylation, ubiquitylation

  • Is a stable type where it is passed down through generations through inheritance.

Hereditary material

1. instructs its own replication

2. stably passed on to its progeny

• through readers and writers the instructs are passed to daughter nucleosomes

Consequence of epigenetic inheritance vs. genetic inheritance (FIX)

• a loss of a nucleosome edit, such as methylation, can lead to an oncogenic cell

• Defective epigenetics can lead to cancer

• When regulation is blocked, an oncogenic cell is made (which can become cancerous)

• however, the epigenome isi dymanic and there can be dynamic changes w/o inheritance that are stable