Activity 6 (Week 10)

RNA Pol 1

Connect fragments, removing primers (same function as in prokaryotes)

• Ribosomal RNA genes

RNA Pol II

Transcribe polypeptide coding genes into mRNA (12 subunits)

• mRNA and some snRNA genes

RNA Pol III

Transcribes tRNA, 5S RNA (subunit), snRNA (splicing)

Transcription Process (1)

Promoter Recognition

Transcription Process (2)

Transcription Initiation

Transcription Process (3)

Elongation

Transcription Process (4)

Termination

Transcription Start Site

DNA Sequences at Eukaryotic Promoters

• +1

• Usually A or G

TATA Box

DNA Sequences at Eukaryotic Promoters

• -30

• TATAAA

• Bound by TATA Binding Protein (TBP)

Downstream Promoter Element (DPE)

DNA Sequences at Eukaryotic Promoters

• +28 to +33

• Bound by TAF6 and TAF9

CAAT Box

DNA Sequences at Eukaryotic Promoters

• About -80

• GGCCAATCT

• Bound by the CAAT box transcription factor (CTF)

GC Element

DNA Sequences at Eukaryotic Promoters

• Somewhere between -200 AND +1 (NO precise location)

• Bound by TF SP1

General Transcription Factors

are REQUIRED to form the RNA pol II complex

Non-general Transcription Factors

are NOT REQUIRED, they just help RNA pol II find TATA box

TFIID

• TAF + TBP

• Binds to TATA Box

  • Could be accompanied by other non-general factors to other parts of the promoter

TFIIA

• Stabilizes TBP binding to DNA and prevents repressor binding

TFIIB

Helps position RNA polymerase II correctly at the start site

TFIIF

• Escorts RNA polymerase II to the promoter

TFIIE

• Recruits the TFIIH and regulates its activity

TFIIH

• Unwinds DNA (helicase) and phosphorylates RNA polymerase II CTD to start transcription

Poised State

Phosphorylation of RNA Pol II

• RNA Pol II pre-initiation complex is NOT phosphorylated

• Complex is formed but not transcription has occurred yet

Paused State

Phosphorylation of RNA Pol II

• Recruits mRNA processing machinery

• CDK-7 phosphorylates Serine 5 in C-Terminal domain of RNA pol II → now in paused state

• CDK-9 phosphorylates Serine 2 → release pol II → transcription

Enhancers

(upstream ~ -500, downstream, or within the gene) work with promoters to regulate transcription = increases transcription

loop over to contact promoters and help initiate and increase transcription

Silencers

sequences that suppresses transcriptions

ex. Pierre Robin Syndrome: cleft palate, reduced lower jaw, posteriorly displaced tongue

Due to mutation in the enhancer for a gene called Sox9

Prevents binding of a TF called Twist1 to the enhancers

pre-mRNA Processing: 5’ End

Called 5’ end cap OR 5’ m7G cap → essential for translation

Importance of 3’ PolyA Tail

• Poly-adenylation is part of transcriptional termination

• Protecting mRNA from degradation (linked to lifespan of mRNA)

  • Facilitating export of mature mRNA across the nuclear membrane to the cytoplasm

Addition of the 5’ mG cap

• Guanylyl transferase (GTase) adds a guanine to the 5’end of the pre-mRNA (additional enzymes then methylate the newly added guanine)

Addition of polyA tail

• Addition of ~200-300 adenines to the 3’end of the pre-mRNA → polyadenylation - necessary for RNA stability and transport into the cytoplasm

RNA Splicing

• Occurs for almost all protein coding genes

• Specific internal regions of transcript are removed and remaining pieces joined together

Mechanism of RNA Splicing

• Requires specific sequences in mRNA

• Cellular machinery called the spliceosome

  • Spliceosome - complex of proteins and snRNA

Steps in Splicing (1)

Cleavage at 5’ splice site and transesterification to join the G of 5’ splice site to branch point = forming “lariat”

Steps in Splicing (2)

Cleavage at 3’ splice site

Steps in Splicing (3)

Joining of exons and degradation of intron

Exons

become part of mature mRNA and encode segments of proteins

Introns

are intervening sequences that separate exons

• during the process of splicing introns are removed and exons are spliced back together

Chromatin

• Consists of DNA and nucleosomes, which are made up of proteins called Histones

• Enables packaging DNA to fit in nucleus

• Regulates DNA replication, transcription and mRNA processing

• Half of the protein content of chromosomes are histone proteins

Histones

Nucleosome = DNA + Histone Octamer(core): 2 molecules of each of H2A, H2B, H3, and H4

High in positively charged amino acids (Lysine, Arginine, and Histidine) = can bind tightly to negatively charged DNA

Heterochromatin

Densely compacted and inhibits transcription

• “closed” or “silent” chromatin

Euchromatin

loosely compacted and is accessible to transcriptional machinery

• “open” chromatin

Writers

Chromatin Modifiers - Types of Enzymes

• Add chemical groups

Erasers

Chromatin Modifiers - Types of Enzymes

• Remove chemical groups

Readers

Chromatin Modifiers - Types of Enzymes

• Proteins that recognize the modified histones

Chromatin Remodelers

• enzymatic complexes that can move nucleosomes around to change compaction state of chromatin (use ATP for energy)

Nucleosome sliding

• Sliding the nucleosome will expose the enhancer or promoter element = allowing TFs to bind

Chromatin modifiers

• chemically modify histones by adding or removing chemical groups, called post-translation histone modifications (histone PTMs)

• Acetyle, methyl and other chemical groups at the specific amino acids in the amino terminal “histone tails”

Active transcription

Lysines K9 and K27 acetylated = Neutralizes the negative charge DNA and relaxes the tight hold of nucleosome on DNA

“Silent” no transcription

Lysines K9 and K27 methylated

DNA Methylation

• does not affect nucleosome and DNA interactions, but instead creates binding sites for regulatory proteins

• Addition of methyl groups is accomplished by histone methyltransferases (HMTs)

H3K9 tri/di-methylation

Constitutive heterochromatin = ALWAYS “closed”

H3K27 tri/di-methylation

• Faculative heterochromatin = Switches between hetero and euchromatin

H3K4 tri-methylation

Active promoters in euchromatin