Lecture 18

Question 1

PIC: Pre initiation complex: general transcription factors and RNAP II assemble at a euk promoter

Answer 1

concept: to regulate transcription, almost all happens at initiation phase (big thing in reg is “when”)
RNA pol core enz + general transcription factors (don’t need to know names) form complex that does what sigma factor does in bacteria
TATA binding protein is a key subunit of TFIID
90% of euk promoters do not have tata seq, but still need tbp
seq in euk genomes not highly conserved
all diff complexes assembled in stepwise process
catalytic part comes in quite late. even once assembled, not enough to get transcription
TBP introduces a 45 deg bend into the double helix, locally untwisting the DNA. will cause to untwist, help open and enable others to get in (gen transcription factors)
TBP is a universal GTF, required for RNAP I, II, and III initiation

Question 2

What do enhancer sequences do?

Answer 2

Regulate the activity of core promoters
semi conserved sequences (Sometimes have tata box)
enhancers are sequences that can be upstream of gene, in an intron, can go in either direction in terms of sense, can b eon either strand
in euk, sites where other transcription factors (not general ones- specific) will bind to other specific enhancers
without these, not activated, nothing happens
complex without enhancers = no transcription
enhancers: short sequences proteins bind to
some only expressed in muscle cells, etc.
“master gene” can define certain cell fate
these can be away from where transcription starts
single gene will often have a whole bunch, each binding to diff transcription factors
specific enhancer sequences bind different “activator” transcription factors
each gene has a different array of enhancer sequences

Question 3

Sets of enhancer sequences bind different combinations of activators to give modular control over each gene’s promoter

Answer 3

diff species have the same transcription factors but rearranged, may be where our differences come from
regulatory elements differ us from a mouse
diff transcription factors, diff combinations, diff genes, diff levels

Question 4

Enhancer sequences regulate the activity of core promoter

Answer 4

must talk to core machinery
dna can actually loop around to bring into contact
probably tons of different transcriptional activating proteins
each must know how to talk to part of machinery
part would have to specifically recognize proteins too
have mediator complex for that

Question 5

Mediator

Answer 5

many activators regulate transcription by interacting with mediator, a huge protein complex of over 20 subunits
Mediator is required for efficient transcription from all RNAP II promoters. Acidic region that mediator complex recognizes
helps bring together specific transcription factors with machinery
mediator essential- otherwise get little transcription
sits in between activator protein and RNAPII

Question 6

Heterochromatin and euchromatin

Answer 6

regions of repressed and activated transcription

dna not just free- wrapped around histone proteins. How do you access DNA when you want to transcribe a gene? get stuff into opened up form to transcribe genes

heterochromatin:
transcriptionally inactive/turned off. condensed chromatin. heterochromatin binds to nuclear envelope

euchromatin: dispersed or extended chromatin

stuff not uniform around the cell: where it is is not regulated. How do you get stuff into opened up form to actually transcribe genes?

Question 7

Histones

Answer 7

4 different proteins, for octamers, positive charges to bind to DNA
also have tails (N term tail)

Question 8

Transcription initiation is regulated by DNA accessibility

Answer 8

histone remodeling complexes
promoter sequence hidden, need to move these guys to get to promoter
takes a lot of energy; motors that use energy of ATP hydrolysis to shove nucleosomes back and forth on DNA. these enzymes resemble helicases, but lack the ability to directly unwind DNA
remodeling complexes have motors that hydrolyze atp to get energy for this; constantly have to move these back and forth. this is essential
histone remodeling complexes hydrolyze atp to get energy to move nucleosomes back and forth to expose/cover up promoter sequence

Question 9

The structure and activity of chromatin is controlled by post-translational modifications (“marks”)

Answer 9

Writer enzymes add marks to chromatin. Eraser enzymes remove these marks. Marks can be placed on histones OR dna.
To produce biological read out, reader proteins specifically bind to the histone modification.
leads to chromatin condensation, chromatin den condensation, transcription activator binding, etc.
basically regulating gene expression based on reading out these marks

some of these marks can be passed down through parents
writers and erasers put on and take off marks
what do these marks mean? no universal rules

modify either dna or histone proteins and enables genes to be turned on and off at certain times
often states are very stable

Question 10

Some enzymes that write and erase chromatin marks

Answer 10

DNA:
-write: DNA methyltransferases
Proteins:
-write: histone acetyltransferases (HATs)
-erase: histone deacetylases (HDACs)
-write: histone methyltransferases (HMTs)
-erase: lysine demethylases (LSDs)
Also: histone kinases, histone ubiquitin ligases…

Question 11

The structure and activity chromatin is controlled by post-translational modifications - Covalent modification of DNA

Answer 11

-methylation (cytosine C-5 in Cpg dinucleotides)
–enzymes: DNA methyltransferases
–substrates: DNA (CpG) and SAM
only does this to specific cytosines. cytosine dinucleotides?
specifically enriched around 5’ end of genes where regulated. in mammals a huge portion are regulated (60-90%)
5th base in dna is putting methyl group on 5C of cytosine
will not affect base pairing. sticks into major groove of DNA, prevents some transcription factors from binding - generally shuts off transcription

Question 12

The structure and activity chromatin is controlled by post-translational modifications - Covalent modification of Histones

Answer 12

-phosphorylation (Ser, thr)
-acetylation (lys)
-methylation (arg, lys) (no direct effect for methylation)
-ubiquitination
1st three: note that phosphorylation adds a negative charge, while acetylation neutralizes a positive charge. methyl group does not add a charge. Similar to ubiq.
Directly control charge of histones. modifications often made on those residues. make it neutral by adding an acyl group. make it bind to dna less tightly; help open up (activation of transcription)
direct effects: what it does based on charge
some indirect effects too

Question 13

N-terminal tails of histone proteins are major targets of regulatory modification

Answer 13

Tails have lysines and args that can be modified

each have tons of different modifications

basically should we open stuff or not?

Question 14

Methylation of histone lys and arg residues by Histone methyltransferases (HMTs)

Answer 14

Each one of these can be a different mark that can be interpreted differently
as with DNA methyltransferases, the methyl donor substrate is SAM (S-adenosylmethionine)
get an idea of potential complexity of this system

Question 15

Complicated patterns of histone tail methylation are associated with different patterns of gene activity

Answer 15

monomethylation= enhancer

but two or three will turn off

similar to ubiquitin code

(one = promoter or activation, two = heterochromatin or repression)

Question 16

In general:

Answer 16

active genes have heavily acetylated histones
(turn gene on: lots of acetylation helps open chromatin and recruit things)

Primed genes (Inactive genes in an activatable state) have less-heavily acetylated histones (int step of acetylation)

Heterochromatin: silenced genes are not very heavily acetylated, but are heavily methylated on dna AND histones (heterochromatin = basically totally silenced. like X chromosomes)

Question 17

Transcription Initiation is regulated by DNA accessibility through chromatin structure

Answer 17

• histone acetyl transferases (HATs) acetylate lysines, especially on the N terminal tails of histones.
• many different proteins, including remodeling factors, bind to acetylated lysines and other modified residues on histones

2 ways to turn a gene one
1) binding of enhancers through the mediator
2) transcription factors will bind to these enhancers, then will recruit HATs part of transcription factor complex
move nucleosomes around, open DNA, etc.
often have both of these methods to turn genes on

Question 18

Transcription elongation: phosphorylation of the RNAP II CTD allow the polymerase to leave the promoter

Answer 18

52 repeats of the sequence YSPTSPS

proline- rigid ring structure
nothing hydrophobic in here. all hydrophilic
no way to fold
ser and thr can be sites of phosphorylation

RNAP II C-terminal domain (CTD) is a floppy, intrinsically disordered part of the RNAP II enzyme

initiation to elongation phase has to do with pol II
phos of this C term domain. is seen as a signal to go from initiation phase to elongation phase

release it: now doing active transcription

phosphorylate the hydrophilic, non folding (b/c of proline’s rigid ring structure) CTD (ser or thr) and this is seen as signal to go from initiation phase to elongation phase

Question 19

Histones are acetylated, displaced, and repositioned during transcriptional elongation

Answer 19

Once the PIC is assembled, the C terminal domain (CTD) of the RNAP II is phosphorylated many times…
allowing the elongation complex to bind and…
the elongation contains a histone acetyl transferase that helps that helps to displace the nucleosomes
as it moves, pol II leaves a “trail of breadcrumbs” as it transcribes the gene

need to move nucleosomes stuck in front: elongation has a whole bunch of proteins, binds to phos tail, has histone acetyl transferases that can move ahead and displace things

Question 20

Eukaryotic mRNA processing

Answer 20

-5’ cap addition
-3’ polyadenylation (poly a tail)
-splicing, to remove introns
several steps to process mRNA to make it fully mature
not really a stepwise process
this stuff happens during elongation

Question 21

The 5’ end of the nascent mRNA is CAPPED by 7-methylguanosine

Answer 21

capping enzyme done this in three steps:

1) hydrolysis removes 5’ gamma phosphate from pre-mRNA
2) guanylation of pre-mRNA through 5-3’ triphosphate bridge (PPi released)
3) methylation of the guanosine base at position N7. Methyl donor is SAM

make 5-5 phosphodiester bond
DNA ligase also has 5-5 linkage
methylate in position that’s not gonna affect the base pairing

capping occurs during elongation. capping enzyme binds to the RNAP II phosphorylated CTD
eukaryotes only! bacterial mRNA has 5’ PPP

C term domain signal to go into elongation phase, and brings along other proteins

put 7 methylguanosine on 5’

Question 22

The 3’ end of the nascent mRNA is poly adenylated

Answer 22

heterogenus

have cleavage signal on 3’ end of nascent RNA chain
cleavage by specific endonuclease, addition of tail by poly A polymerase
get polyadenylated mRNA precursor

euk: initially make various lengths, will cut it off upstream of coding part, cleavage signal is specific, have 3’ end, then enzyme just comes in and adds a bunch of a’s. maybe makes more stable, maybe enables them to get transported outside the nucleus.

virtually all euk genes poly adenylated. happens in fact too but not necessarily typical.

Question 23

Most eukaryotic mRNA transcripts are spliced

Answer 23

this modification is the weirdest

introns are removed, exons remain

DNA coding region for the actual mRNA is way bigger. meant you had to get rid of stuff from within

back then, this was a shock. often had 5’ and 3’ ends already
stuff your’e getting rid of had to come from somewhere in the middle

Question 24

ovalbumin gene and its mRNA

Answer 24

parts of DNA that had nothing to pair with would form loops

were able to find where the 5’ end was
could figure out how many chunks were removed from the gene

demonstrated that euk genes were built this way

Question 25

Some transcripts exhibit tissue-specific splicing

Answer 25

introns in all orgs, but in bacteria mostly don’t have them

found in common ancestor and then bacteria got rid of them

introns are probably how genes evolved

elks don’t care as much about having extra dna, we can use this to make isoforms of proteins (proteins that are slightly alike)

Question 26

Two nt’s at the beginning and end of an intron are invariant

Answer 26

most introns are removed through the lariat mechanism

conserved nucleotides

look at what nucleotide is in each position and the percent of the time it occurs

particular nucleotides tell where to splice

also have 2’ hydroxyl coming off in the middle, which will give an int

get a lot more proteins that are related

thought we’re more complex, we have more genes

BUT human has the same number of genes as a fruit fly

get more bang for our buck b/c of alternative splicing, etc

Intron: GU to AG

Question 27

The lariat mechanism

Answer 27

a residue whose 5’ hydroxyl will attack at 5’ pG

gives cyclic intermediate

then OH attacks other splice site

get the two eons together

leftover introns can be important, it turns out. can encode other reg RNAs

Question 28

mRNA splicing is carried out by the spliceosome, composed of snRNPs (small nuclear ribonucleoproteins- snurp)

Answer 28

U7 snRNP- controls processing of 3’ end of the mRNAs encoding histone proteins

the adjective small is misleading. the RNA components are small compared to some other RNAs, but this is an enormous (45s) complex, one of the biggest in the cell

small RNAs (about 5 diff ones)
and a ton of proteins
together, form spliceosome
holding onto these pieces and keeping them aligned
has a lot of base pairing
some orgs can do splicing without the spliceosome

Question 29

Group 1 introns are autocatalytic: they splice themselves

Answer 29

Certain eukaryotic ribosomal RNAs can do this. this was the first example of RNA catalysis. it was utterly unexpected. many many other examples have since been discovered.

first example of enzyme with only rna and no protein. add free guanosine, can still do this. not just being used for energy. free g attacks that first site.