BIO367 - Concordia University

Dr.Andrew Wieczorek - Winter 2025

What are the characteristics of RNAPI?

found in nucleolus

transcribes large precursor to major rRNAs

transcribes one gene

What are the characteristics of RNAPII?

found in nucleoplasm

transcribes precursors to mRNAs (hnRNA + snRNAs)

What are the characteristics of RNAPIII?

found in nucleoplasm

transcribes precursors for 5srRNA and tRNAs

When cloning a plasmid using a single cut site, what is the vector treated with before ligation?

Alkaline phosphate

removes 5’ phosphate that ligase needs to self-ligate back onto itself

When using DNA footprinting to identify different promoters, what would one expect to see from a class I promoter and what are the elements called?

one core element and an upsteam element (UPE) about 100-150bp upstream of transcription initiation

When using DNA footprinting to identify different promoters, what would one expect to see from a class II promoter and what are the elements called?

BRE - binding site for TFIIB (-37 to -32)

TATA - binding site for TBP (-26 to -31)

Inr - binding site for TFIID (-2 to +4)

DCE - binding site for TFIID (+6 to +33)

MPE (within DCE) - downstream promoter element

MTE (within DCE)

When using DNA footprinting to identify different promoters, what would one expect to see from a class III type I promoter and what are the elements called?

5S rRNA

Box A, intermediate element, Box C

When using DNA footprinting to identify different promoters, what would one expect to see from a class III type II promoter and what are the elements called?

tRNA

Box A, Box B

When using DNA footprinting to identify different promoters, what would one expect to see from a class III type III promoter and what are the elements called?

snRNA gene

DSE (distal sequence element), PSE (proximal sequence element), TATA box

What is the order of binding DNA to create the preinitiation complex.

TFIID, TFIIA, TFIIB, RNA Polymerase II, TFIIF, TFIIE, TFIIH

Where do the TBP/TFIID bind DNA?

The minor groove

What is TFIID

TBP (TATA binding protein) + TAFIIs

Why are TAFIIs important?

They bind the Inr

They bind the DPE

They bind to other factors important for transcription

What is the last TF to join the preinitiation complex?

TFIIH

What is the function of TFIIH?

It is a helicase - unwinds DNA at transcription site

TFIIH phosphorylates the C-terminal domain of RNAPII and is stimulated by the presence of TFIIE

What is the function of TFIID

Has TBP, first TF in preinitiation complex

What is the function of TFIIF?

Aids in polymerase recruitment - does not bind have binding site for DNA

Does directionality matter in enhancer sequences?

no

Where does TFIIH phosphorylate RNAPII?

C-Terminal Domain (CTD)Wha

What does TFIIS do?

stimulates stabile transcription elongation

Proof-reading activity of transcript

What does TFIIIC do?

It binds Box A and Box B

Recruits RNAPIII

Why is the TATA box important

gives location of transcription start site + transcription efficiency

Why is the large intron of most class II genes important?

There is an enhancer within this intron that is needed to generate mRNA

Is the TATA box an enhancer?

no, it is a promoter

Where on DNA does the TBP bind?

The minor groove

In what situations are TAFIIs more important for transcription

more important in genes with a Inr or a DPE

less important in genes with just a single TATA box

Is TBP still required for transcription even if there is no TATA box?

yes, it is within TFIID which also has other TFs which need to be assembled and present for transcription to occur

TBP may not bind to DNA, but other TFs are bound to it which may interact with other TFs that are bound to DNA

What is the order of TF binding in type III promoter regions?

TFIIIC, TFIIIB (contains TBP), Polymerase (TFIIIC falls off during transcription as to not block polymerase)

What are activators?

Gene specific transcription factors

What are the 3 domains of an activator?

DNA binding domains

Transcription Activation Domains

Dimerization Domains (sometimes not needed)W

What is the charge of DNA?

negative

What is SP1 and what does SP1 bind to?

SP1 is a C2H2 zinc finger activator for class II promoters

binds TATA-less promoters —> GC boxes

What kind of transcription factor is Gal4?

Activator

What is the role of Galactose in the Gal operon?

Galactose binds Gal80 which relieves repression of Gal4

What does Gal80 do in the Gal operon?

Gal80 is the repressor for Gal4

The different expression pattern of the yeast Gal1 is determined by its gene’s enhancer/UAS region

yes

What was does genetic imprinting say?

the genes expressed by offspring expressed by the methylated gene passed down by paternal mouse

What mechanism does activation occur?

Enhancer functions to bind activators, activators use looping mechanism to recruit general transcription factors and RNAP promoters

What does ‘squelching’ mean?

One activator inhibits another activator from working by competing for the same binding site

Do mediators bind DNA?

no

What is heterochromatin?

dense, silenced DNA

packed tight

What is euchromatin?

loose DNA almost always under transcription

What subunits are the core histone made of?

H2A, H2B, H3, H4

In histones, what is H1?

The linker protein

What is the charge of the histone tails?

Positive - binds to DNA because DNA is negatively charged

In histone modification, what amino acid is modified during acetylation?

Lysine

What is the relationship between activators and histones?

Histone and activators compete

Activators act as both activators and antirepressors

What does histone acetylation do to the interaction between histones and DNA?

weakens the interactions between DNA and the histone

What does histone deacetylation do to the interaction between histones and DNA?

strengthens the interactions between DNA and the histone

What protein complexes are required for chromatin modifications for gene activation?

Histone Acetyltransferases (HATS)

Chromatin Remodeling Factors (SWI/SNF)

Where does Rap1p bind on DNA?

Rap1p binds to the telomeres

What class of genes does the spliceosome act on?

Class II genes

The first 2 bases of an intron are

GU

The last 2 bases of an intron are

AG

Can the branch point ‘A’ in an intron be any A within the intron?

No, requires consensus sequence

What are spliceosomes made of?

Snurps

U1, U2, U4, U5, U6

In the spliceosome, what does U1 recognize and bind?

5’ splice site of intron

In the spliceosome, what does U2 recognize and bind?

The intron branching point of intron

What is U1 nudged off by in the process of the spliceosome?

U6 (associated with U5)

In the spliceosome, where does U5 recognize and bind?

3’ splice site of intron

Is U1 in the spliceosome sequence specific?

yes, mutation causes lack of splicing

countermutation to create complementarity yields splicing

Is U6 in the spliceosome sequence specific?

yes, mutation causes lack of splicing

needs UGU on pre-mRNA to pair with U6 ACA

Is U5 in the spliceosome sequence specific?

No

Is U2 in the spliceosome sequence specific?

yes

What does the mechanism of group I self-splicing introns look like?

Sequence of intron made such that it adopts a tertiary structure and a nucleophilic attack occurs which cleaves the intron without the need for spliceosome

What does the mechanism of group II self-splicing introns look like?

-OH group on branch point adenine performs same double nucleophilic attack to splice itself without need for spliceosome

creates lariat structure

What is the difference between cap 0, cap 1, and cap 2

Cap 0 has no methyl groups, cap 1 has 1 methyl group on the first nucleotide, and cap 2 has a methyl group on both the 1st and 2nd nucleotide

What is the order of capping for cap 2

RNA triphosphate clips gamma-phosphate (leaves just aB phosphates)

Guanylyl transferase attaches GMP (reestablishes 3 phosphates on Guanine aBa)

methyl transferase adds Met to guanine cap

methyl transferase adds Met to guanine cap

What processes are 5’ caps required for?

Intron removal

mRNA transport out of nucleus

translation by ribosomes (CAP binding protein)

mRNA stability (protect mRNA from RNAse)

What does the 3’ poly-A tail do?

protects mRNA from RNAse

What does the GU/U element at the 3’ end of mRNA after the polyadenylation signal do?

GU/U element is bound by CFs (cleavage factors) to cleave site for polyadenylation

What does the polyadenylation signal look like/do?

AAUAAA + GU/U element after

marks site for poly-A tail

What is the rate of polyA degradation determined by?

signal in 3’ UTR

How does the CoTC site contribute to transcription termination?

CoTC region forms a transcript that cleaves itself on one end

The cleaved end is bound by Xrn2 exonuclease which then chases down polymerase and knocks it off the mRNA

What does DICER do?

enzyme that cleaves dsRNA into siRNA

What is rho dependent termination?

rho binds to polymerase and waits for rho dependent termination site — then rho dissociates transcript from polymerase

What is a rho dependent termination site?

inverted repeat

What is the mechanism rho independent termination

what does β-galactosidase do?

Breaks lactose into galactose and glucose

What is the inducer of the lac operon?

Allolactose

What gene in the lac operon codes for cAMP?

cyaA

In the lac operon, what is the effect of high glucose on cAMP?

When glucose is present, cAMP production is low

Where does the CAP-cAMP complex bind to RNA polymerase?

α-subunit