Biol 304 Discussion 8

Is the TATAAT sequence always centered at -10 nucleotides from the start?

Usually, but not always

On which strand is the TATAAT sequence found?

Message (+) strand

How does RNA polymerase bind first during transcription?

Guided by sigma factors, it binds to a promotor on the template strand to synthesize the message strand

On which strand is RNA polymerase found when it is transcribing DNA?

Template (-) strand

Where would you expect DNA to be melted more easily: at -10 or at -35?

-10 because it’s AT rich (less H bonds)

Here is the consensus of the -10 region of a promoter: T80A95T45A60A50T96. If the -10 sequence is CACCAC, will transcription still occur from that promoter?

No since this is a highly conserved region and the given sequence is very different than it should be

When the % of alignment gets close to 100%, what does it mean?

That specific nucleostides are more and more likely to be present at a given position.
Highly conserved

Highly important

What are 4 conserved elements in a bacterial promoter that were discussed?

TTGACA consensus centered @ -35

spacing element (16-18bp) between TTGACA and TATAAT @ -35 to -10

TATAAT consensus centered @ -10

start point @ +1 (90% of time purine)

Does the sequence between -10 & -35 affect levels of transcription?

Does the distance between -10 & -35 affect levels of transcription?

What type of mutation would affect the level of transcription in the region from -35 to -10?

What type of mutation would not affect the level of transcription in the region from -35 to -10?

No;

Yes;

insert/delete;

Base substitute

Which sequence allows RNA pol to initially recognize a promoter? Which sequence provides an unwinding domain?

-35 (TTGACA) (sigma ⁷⁰ subunit);

-10 (TATAAT) (Beta’ subunit)

What is the name for the structure formed when sigma binds to RNA Pol?

RNA polymerase holoenzyme

When is the sigma factor usually released from RNA Pol?

after initiation (when it clears the promoter)

Can the sigma factor stay associated with RNA Pol?

Yes, but usually releases after promotor clearance

What is the effect of the sigma factor on polymerase binding?

• dictates promoter specificity

• helps core enzyme bind to a specific site

To what sites does RNA Pol bind when it is not bound to a sigma factor?

loose-binding sites which are randomly chosen (bc it falls off)

What are loose and specific binding sites?

loose: when RNA Pol has bound randomely since no sigma factor is bound

specific: promoter regions & specific genes found by sigma factor bound to RNA pol

Can different sigma factors recognize different promoter sequences?

Yes

What is different about RNA Pol when a specific sigma factor is bound to it?

• stable and tight binding to promoter region

• it will recognize a specific sequence

What might be the effect if there is a mutation in the DNA binding domain of the sigma factor?

Might not bind -35 or -10

Will not be able to do specific binding (only loose binding)

Is the sigma factor found in eukaryotes?

No

What is one example of sigma factors being "switched?"

When bacteria experience heat shock they will switch from standard sigma 70 factor → heat shock sigma 32 factor

What happens to sigma proteins in the heat shock response?

sigma 70 is unbound (comes off pol) and sigma 32 comes on which activates the heat shock response and expresses chaperone proteins

Which RNA Pol transcribes genes in mitochondria & chloroplasts?

There are special RNA polymerases for each and they have their own.

What is the CTD?

• Carboxyl / C- terminal domain

• has many repeats of the consensus sequence

Which RNA Pol has a CTD?

DNA Pol II

What sequence is specific to the CTD?

YSPTSPS (consensus sequence)

What can happen to amino acids containing -OH functional groups serine, threonine, tyrosine?

phosphorylation

What is the role of upstream binding factor (UBF) in RNA pol I gene expression?

regulates rRNA gene expression by increasing efficiency of initiation

If boxes such as boxA, boxB, and boxC are found in many eukaryotes are they said to be conserved or have a consensus sequence?

• conserved (same across many species)

• consensus just tells us % chance sequence will contain certain nucleotides (as it usually contains them)

Which RNA Pol transcribes RNA that will be translated into proteins?

RNA Pol II

What is the core promoter?

• Short, minimal, around the startpoint (+1) in DNA

• helps w/ transcription initiation

Can transcription initiation occur when only the core promoter is present? What is TBP?

• Yes

• TBP = "TATA-binding protein"

  • binds to DNA's minor groove at the TATA box

Are all eukaryotic promoters located before transcription start point? How does this compare to prokaryotic promoters?

No (can be found anywhere near startpoint)

Prokaryotic promoters are always upstream

What is the general purpose of enhancers?

• Increase transcription rates by increasing RNA pol to promoter binding efficiency

• stimulate transcription by recruiting activators & general binding proteins

• Stabilize RNA Poly

What allows more activators to be recruited?

enhancers

What is one characteristic that distinguishes promoters from enhancers?

• Promoters: near & fixed position from startpoint

• Enhancers: vary in distance & can be located up or downstream

What is one difference between enhancers and activators?

Enhancers: DNA that binds activators (proteins), cis-acting

Activators: proteins, trans-acting

What DNA sequence might prevent enhancer-promoter interaction?

insulator sequences

What is cooperative binding?

• When a protein changes to connect more easily to other molecules when one is already bound first

  • so after 1st binding, 2nd is easier, etc...

When an enhancer is acting on a promoter, is a physical connection required between these 2 sites?

yes

What are CpG islands?

• DNA regions with high C & G concentration in regulatory regions

There are a lot of CpG islands before housekeeping genes. What happens when CpG islands are methylated? What hapens when CpG islands are not methylated?

• methylated: gene transcription turned off

• unmethylated: gene transcription turned on

Can enhancers be found in introns & other DNA sequences that are not expressed?

Yes

central dogma

dna → rna → protein

dna → dna (replication)

dna → rna (transcription)

rna → dna (reverse transcription)

rna → protein (translation)

sense strand synonymous for

• non template

• coding

• message

• (+)

• = mRNA sequence w/ T replaced w/ U

anti-sense strand synonymous for

• template

• non-coding

• non-message

• (-)

• complementary to sense

prokaryotic replication vs transcription (table)

• template

• product

• # strands synthesized

• major enzyme

• types of nucleotides used

• enzyme binding site

• start site

• region of synthesis

• primer needed

• proofreading

• reading direction

• direction of synthesis

• bubble size

• directionality of syn

prokaryotic transcription overview (diagram)

how many RNA polymerase in prokaryotes?

one

what core enzyme makes up prokaryotic RNA pol? + what type of binding for initiation and releasing after promoter clearance?

α₂ββ'ω

sigma binding

w= omega

rna pol holoenzyme

α₂ββ'ωσ

rna pol subunits + function

consensus sequence

what does the subscript mean?

calculated order of most frequent residues found at each position in a sequence alignment ; the nts are known

subscript = % that a specific nucleotide occurs in all known examples

conserved sequence

similar or identical sequences which occur in DNA

occur across species

TTGACA consensus

centered @ -35

spaced conserved between species

distance between TTGACA and TATAAT = 16-18 bases

TATAAT consensus

centered around -10

prokaryotic promoted diagram

nucleotide changes to consensus sequence affect what?

rate of transcription

incrase/decrease # nucleotides between two consensus sequence affects ___

rate of transcription

nucleotide changes between two consensus sequences will ___

NOT affect transcription (substitutions fine)

• insertion/deletion WILL impact!

eukaryotic transcription vs prokaryotic transcription (TABLE)

• promoter

• enzyme binding to promoter

• name of TAATAT consensus

• location of TAATAT consensus

• # of RNA polys

• proteins needed for intiation

• does RNA pol bind directly to promoter?

3 classes of eurkaryotic rna pol + functions

• rna pol I

  • synthesis of rRNA (ribosomal rna)

• rna pol II

  • synth of mRNA

• rna pol III (3 types)

  • synth multiple types of rna

each rna pol have same promoters?

NO

cis-acting elements

dna sequences

(intrinsic/within)

affect same molecule

ex: promoter

trans-acting factor

proteins that bind to cis

(extrinsic/external)

affect different molecule

ex: transcription factors

rna pol I promoter

why must CTD tail be phosphorylated on S and T residues for RNA pol?

to

rna pol II promoter

rna pol III promoters

rna pol III type 1 promoter

rna pol III: type 2 promoter

rna pol III: type 3 promoter

eukaryotic rna pol and promoters (TABLE)

RNA pol I | RNA pol II | RNA pol III (Type 1, 2, 3)

• promoter type

• has CTD?

• location of transcription

• effect of alpha-amatin on pol

• products

• core promoter

• other cis-acting elements

• trans-acting factors

• location of TATA-binding protein (TBP)

enhancer def

• stimulates what?

• cis or trans?

• far or near start point?

dna seq which can greatly increase transcription rates by increasing efficiency by which rna pol binds to a promoter

• stimulates transcription initiation

• cis-acting elements

  • farther away from start point (up or downstream)

DIFFERENCES promoters vs enhancers (table)

• distance from start point

• works if they are invered

• found up or downstream

• many repeats

• works when diff parts are ‘mixed’

• rna pol binding

SIMILARITIES between promoters and enhancers

• dna seq that serve as protein binding sites

• have consensus sequences

• proteins bound to them interact w. transcription machinery

• both can recruit TFs that remodel chromatin