MBB222 - Lecture 28: Eukaryotic Transcription and processing

why is Eukaryotic transcription much more complex than prokaryotic transcription

• have a nucleus → transcription and translation can’t occur at the same time

• have chromatin → transcription machinery must work around nucleosomes as chromatin packaging represses transcription

• have more complex processing of mRNA

• have 3 RNA polymerase - more complex activation of expression

eukaryotic genes are transcribed as what kind of messages

transcribed as monocistronic messages

what is a monocistronic message

mRNA transcript that carries the genetic code to build one single protein

where is RNA polymerase I / II / III found

RNA poly 1 → nucleolus

RNA poly 2 → nucleoplasm

RNA poly 3 → nucleoplasm

what does alpha-amanitin do

stops RNA polymerase 2 during elongation phase of transcription

how can alpha-amanitin be used to determine which RNA polymerase transcribes a given RNA in vivo

by testing it’s sensitivity to alpha-amanitin

RNAP 1 → insensitive (not affected)

RNAP 2 → strongly inhibits

RNAP 3 → is inhibited by strong concentrations

Which promoter regions recruit which RNA polymerase

RNAP 1 → recognized by core promoter elements + upstream promoter elements

RNAP 2 → recognized by upstream elements (i.e. TATA box) + Inr (initiator) + DPE (downstream promoter elements)

RNAP 3 → has unique promoters (Box A / Box B / Box C) - often downstream of the actual gene sequence

why is mRNA the key to cell specialization and adaptation

acts as a temporary instruction carrier that directs which proteins a cell builds → allows different cell to use the same DNA library to create unique futures

what does the synthesis of mRNA require

• DNA template

• RNA polymerase

• ribonucleotide

• ATP / CTP / GTP / UTP

why are there many different messenger RNA (mRNA) molecules in a cell

each one carries a unique set of instructions to make a specific protein

what are cellular proteins derived from

mRNA

what makes mRNA

RNA polymerase II (also makes snRNA → small nuclear RNA)

what is required to initiate the formation of the open complex - the start of RNA synthesis

the chronological loading of transcription factor proteins to RNA polymerase II promoters

what is the sequential loading order of factor proteins to RNA polymerase II promoters

TFIID → binds first to core promoter by TBP (TATA binding protein - recognizes the TATA box) - bends the DNA

TFIIA → binds to stabilize the TFIID-DNA complex

TFIIB → binds to TBP and flanking DNA sequence - sets the right direction / start site for transcription

TFIIF / RNA polymerase II → are together as a pre-formed complex - is recruited to the promoter through the interaction with TFIIB

TFIIE → joins the complex after RNA polymerase II - helps recruit TFIIH

TFIIH → binds last to the pre-initiation complex - helicase subunits unwind the DNA - kinase subunit phosphorylates the C - terminal domain of RNA polymerase II to start transcription

which amino acid are in TATA binding protein (TBP)

phenylalanine (Phe)

which groove does the Phe residues bind to

minor groove

How does TBP allow RNAP II to bind

• binds to the minor groove

• binding induces a large conformational change in the DNA - bends it - spreads the minor groove

• distorting the DNA can signla an active promoter → allowing RNAP II to bind

why does TBP only bind to the minor groove

its unique saddle - shaped tertiary structure is geometrically complementary to the flattened and widened minor groove of AT - rich sequences

why is positive regulation more prominent in eukaryotic genomes

the default state of eukaryotic DNA is tightly packed and inactive → requires specific activator proteins to turn genes on

why is negative regulation by protein repressors is not as crucial as it is in prokaryotes

chromatin structure provides a pre-existing repressed state

why does the initiation of transcription require multiple activator

to achieve precise gene control, chromatin remodelling, combinatorial control (where multiple transcription factors work together to control the activity of a gene)

what actions are required in order for an active RNAP II complex to form at the promoter

• histone modifying enzymes and chromatin remodelling proteins → exposes the DNA that will be transcribed

• regulatory proteins that promote transcription

how does activators help the polymerase initiation complex (PIC)

helps in the assembly of PIC by helping recruit RNAP II to the promoter

what are mediator proteins

allow the activators to communicate with RNAP II and other TF (transcription factors)

what are mediator proteins necessary

to overcome the difficulty in binding to DNA that is packaged in chromatin

why are chromatin remodeling complexes and histone acetlases also necessary

in order to move nucleosomes around and allow the transcription factors, polymerases, and activators access to the DNA

how does histone modifications influence TF / polymerases / activators interactions with DNA

changes DNA - histone electrostatic charges (neutralizes the charges on the positive charged lysines) and recruit specific reader proteins

what is the TAP binding site

an enhancer sequence that binds the transcription activator protein (TAP)

describe the steps of eukaryotic transcription initiation

• native chromatin is condensed and tightly wrapped around nucleosomes

• chromatin-remodelling complexes bind to the target region

• regulatory sequences of the DNA become fully accessible → TAP binding site (enhancer) and TBP binding site (promoter) are available

• transcriptional activator protein (TAP) binds directly to its newly exposed enhancer site

• TAP recruits general transcription factors → TFIID + TBP to the TATA box

• RNA polymerase holoenzyme is recruited to the transcription complex to initiate gene expression