BSCI 331: Exam 3 - General

transcription factors generally act at one of two types of gene regulatory regions. what are they called?

promotor or enhancer

promoter

• region where RNA polymerase and general transcription factors assemble

• always a short distance “upstream” of the 5’ end of the gene

• region required for transcription initiation, but may only provide low level of transcription

• may be gene-specific (won’t work if it is put next to a different gene), and its orientation may be important

enhancer

• independent region outside the promoter

• may be very far away from promotor (up to 10s of kb) and may be upstream of the gene, downstream, or within the gene

• region cannot drive transcription on its own, but dramatically increases transcription initiation from its corresponding promoter

• generally position- and orientation-independent, can work with a heterologous (where enhancers can work with any promoter) promoter (promoter from a different gene)

  • “enhancer trap”: minimal basic promoter “upstream” of promoter gene, takes random genes from genome and insert inside plasmid

• not dependent on location

Major groove

• wide groove of DNA (~22 Å) that exposes distinct chemical patterns of base pairs (also able to differentiate base pairings because has methyl groups, H acceptors and H donors involved)

• primary site where transcription factors bind and read DNA sequence (alpha helices bind here)

Minor groove

• narrow groove (~12 Å) with less chemical information

• less commonly used for sequence-specific protein binding

• easily able to detect base pairings since only have hydrogens that interact with one another to transcribe dna transcription

why do transcription factors bind the major groove?

provides more accessible and distinguishable chemical information for base pair recognition without unwinding DNA

Helix-turn-helix (HTH)

• dna-binding motif with two alpha helices

• one helix inserts into the major groove to interact with base pairs that is one full turn away and/or from the dna helix

recognition helix

part of HTH motif that directly contacts DNA bases via hydrogen bonds in the major groove

zinc fingers

• stabilized by zinc ions

• finger-like domains insert into major groove where each finger recognizes ~3 base pairs

leucine zipper

• forms coiled-coil structure from hydrophobic interactions when one helix from one subunit corresponds with following helix with it’s subunit that creates this

• enables dimerization region (only bind dna as symmetric dimers!) and binds as dna binding region

helix loop helix (HLH)

• two helices where one is short and other is long that is connected by a long and flexible unstructured loop that allows the motif to fold back and pack onto themselves facing the surface of the protein

• allows dimerization and dna binding regions o form

• creates two separate dimer regions: homer dimer → identical subunits; hetero-dimer → identical recognition sites or helixes but different recognition sequences which allow for an increase in sequence specificity and recognition, including larger diversity and combination

why is dimerization important for dna binding moifs?

increases binding specificity, stabiliy, and expands the number of dna sequences that can be recognized

homodimer

complex formed by two identical transcription factors (ex: AA, BB, CC), identical subunits

heterodimer

complex formed by two different transcription factors (ex: AB, AC, BC)

how many combinations can you make or do with 3 transcription factors or bases of A, B, and C?

6 → AA, BB, CC, AB, AC, BC

why is AB the same as BA?

because the physical complex is identical regardless of order

what is the effect of increasing transcription factors?

leads to exponential increase in possible DNA binding combinations and regulatory diversity

minimal promoter

smallest DNA region that can initiate transcription at low levels, doesn’t highly activate transcription a lot → “enhancer trap”

enhancer location

• enhancer not dependent on location, but rather in position of where promoter is at in DNA to be able to drastically affect transcription effects

• can be upstream, downstream, or within introns; may be thousands of base pairs away and can be randomly put anywhere

enhancer properties

position independent and orientation independent

difference between promoter and enhancer

• promoter is required to start transcription

• enhancer only boosts/enhances transcription process once next to promoter

“upstream”

• direction of promotor that is short distance and is at the 5’ end, direction opposite to RNA polymerase movement and promoter location

• RNA strand gets synthesized from 5’-3’ while DNA gets read from 3’-5’

“downstream”

direction RNA polymerase moves during transcription and includes gene coding region → adds nucleotides along the way

mediator complex

large protein complex that attaches to RNA polymerase and connects TFs at enhancers to RNA poly. at promoter

DNA looping

bending of DNA that allows distant enhancers to interact with promoters by folding → allow better transcription

combinatorial control

regulation of gene expression by multiple transcription factors acting together

additive effect

combined effect equals sum of individual TF effects

synergistic effect

combined effect is much greater than sum of individual effects

antagonistic effect

one transcription factor opposes the effect of another (activator vs. repressor)

Lac operon

bacterial gene system controlling lactose metabolism

Lac repressor

protein that binds operator and blocks transcription in absence of lactose

effect of glucose on CAP

low glucose leads to high cAMP when lactose is present and binds to DNA

CAP (activating protein for catabolite activator)

activator that binds to DNA with cAMP when glucose is absent or low and when lactose is present

effect of lactose on repressor

lactose binds to repressor when glucose absent → causes conformational change to release DNA

when is lac operon ON?

when lactose is present and glucose is absent that initiates transcription

transcription factor functions

• help to unpack chromatin, making gene accessible to RNA polymerase and initiation complex

• control recruitment of RNA polymerase and/or the general transcription factors to the promoter

• may regulate the swich from initiation to elongation → leads to conformational change

• help recruit histone-modifying enzymes to change the local chromatin structure → can shift nucleosomes close to TF to allow transcription to happen or alter position

• bend DNA to allow long distant interactions between gene regulatory regions in 3D space → more access to transcription

• can serve as activators or repressors

repressor

protein that inhibits transcription

mechanisms of repression

blocks RNA polymerase, compete with activators/enzymes, recruit inhibitory enzymes, prevent DNA looping

TF localization control

TFs inactive in cytoplasm but active in nucleus since there is DNA access there

NF-AT and NF-kB

• immune system discovery of TFs

• NF-kB known to be really important TF in all of eukaryotic biology and controls everything

• both held in cytosol in “inactive” state

• post-translational modifications lead to release from cytosol and translocation to the nucleus

• nuclear TF is able to regulate gene transcription

Combinatorial control (gene expression)

multiple TFs work together to regulate a single gene → may involve with high affinity and sequences geting squared

why is combinatorial control important?

allows limited number of transcription factors to generate diverse gene expression patterns

even-skipped (eve) gene

developmental gene expressed in 7 stripes in the embryo → each stripe is associated with 7 of its modules in DNA

why does mutation of even-skipped remove even-numbered segments?

each stripe corresponds to formation of specific body segments

stripe-specific enhancer

DNA module that controls expression in one stripe of the embryo

why can’t a single gradient explain stripe patterns?

gradients produce continuous expression, not sharp on/off boundaries

how are sharp gene expression sripes formed in DNA?

through overlapping gradients of activators and repressors

bicoid

activator transcription factor highest at anterior

giant/Krüppel

repressor transcription factors that define stripe boundaries

what determines position of stripe 2 in even-skipped?

region where activators are high and repressors are low (“just right” zone) → would transcribe for sequence 4 in DNA

why are TFs pre-made in cells?

allows rapid response without waiting for transcription

what is post-transcriptional regulation of TFs?

controlling activity of already-made proteins instead of making new ones → able to modify them

maternal effect genes

genes whose mRNA is deposited into the egg by the mother

maternal mRNA gradients

establishes early positional information in the embryo

why don’t mammals rely on maternal gradients like fruit flies?

mammalian eggs are too small for spatial gradient formation

how do cells sense position in embryos (2 ways)?

1. direct cell-cell contact

2. diffusible signaling molecules or gradients

transcription attenuation

lead to premature termination of transcription due to RNA structure

how does attenuation work?

RNA forms hairpin conformation structure called “stem loop” → interferes with RNA polymerase activity by pausing on DNA → gets released and transcription stops

can attenuation be reversed?

yes! it can be reversed by binding of specific proteins to the RNA structure, allowing RNA polymerase to complete transcription

alternative splicing

• produces different mRNAs from the same gene → leads to different variations of different proteins at the end

• may be regulated by splicing repressors or splicing activators

• sometimes there are mutually exclusive alternative exons

• “splice variants” involved: gene with even just few exons can produce many different mRNAs via alternative splicing → some variants may simply lack one or more exons

exon skipping

specific exon is removed during splicing and doesn’t get contributed to splice out a intron of RNA

mutually exclusive exons

only one exon from a set is included

why is alternative splicing important?

increases protein diversity without increasing gene number

alternative poly A site selection

• produces different mRNA lengths → have more than one included so can be longer or shorter version that can go “upstream” or “downstream”

• affects stability, localization, translation of mRNA

how can one gene produce multiple proteins?

through alternative splicing and poly A site selection

mRNA nuclear export regulation

• allows mature mRNA to exit out of nucleus into cytosol of cell in order for RNA to get translated to turn into protein

• determines whether RNA leaves nucleus for translation

how do viruses like HIV exploit nuclear export?

• would need some proteins to disrupt certain regulation so some full length RNA get processed

• allows some RNA that are not processed to get exported in cytosol to be put into viral molecules

• export unprocessed rna to produce viral genomes

why localize mrna?

to ensure protein is made exactly where needed

mrna stability regulation

• can be determined based on 5’ and poly A 3’ caps where if neither of them are included then it will get immediately degraded because loss of structure

• controls how long mrna persists in cell

iron

• toxic heavy metal

• really good at oxidizing oxygens = REDUCING oxygen

• iron ion oxidizes oxygen in minus one state

ferritin

• iron storage protein

• made and used when there is too much iron that was produced → over excessive

• protects iron from getting damaged

transferrin receptor

• imports iron into cell

• makes fair amount of iron to produce it

• must be made immediately to quickly restore iron to continue rapid response of metabolism

what protein senses iron levels

aconitase

how does aconitase regulate transferrin receptor mRNA (produces iron amount to continue iron metabolism)?

• low iron → binds mRNA → stabilizes it

• high iron → releases → mRNA degraded

why regulate at mRNA stability instead of transcription?

faster response to environmental changes

miRNA

• micro RNA or little RNA that partially base-pairs with mRNA to inhibit translation

• derived from precursors that fold into “hairpin” stem-loop structures → relates to transcription attenuation

• single gene product, folded to short double stranded pieces of protein

RNA induced silencing complex (RISC complex)

• protein complex that uses RNA guide to silence mRNA, gene silencing

• after processing, short double-stranded rna (stem looped structure called miRNA) is generated and associates with a set of proteins to form this complex

• one strand of RNA is degraded and other strand makes base-pairing contacts with an mRNA target

what determines whether mrna is cleaved or just repressed?

based on degree of base pairing where target mRNA may be degraded, or translation may be inhibited

siRNA (small inhibitory rnas)

• mediate process of rna interference → artificially regulate cells and work catalytically

• small rna that perfectly base-pairs and causes mrna cleavage

• associate with slightly different set of proteins to form rna induced transcriptional silencing complex which inhibits gene transcription by modifying chromatin structure

what enzyme processes double stranded rna (dsRNA) into small rnas?

• dicer

• double stranded rna (dsRNA) formed by base pairing between complementary regions of separate rna strands, formed opposite sides of rna

• dsRNA cleaved by dicer nuclease to form short double-stranded RNAs → siRNA (small RNA), gets cut up

translation regulation (5’ end UTR)

untranslated regions (UTRs) structures can block ribosome scanning in 5’-3’ → 5’ UTR RNA structure can allow binding of translation repressor protein that blocks ribosome access

mRNA circularization in translation → what helps it remain maintained during translation in relation to the 5’ and 3’ ends?

interaction between 5’ cap and 3’ poly A-tail (adenylation) that enhances translation

riboswitch

ribosome switch that acts as a on and off switch that alters the RNA structure upon ligand binding → riboswitch structure may use binding of an ion or small molecule to switch between translation “on” and “off” states

eIF2

• initiation factor that delivers initiator met-tRNA (AUG) once binds to GTP → can inhibit global protein synthesis

• at first is inactive because bounded to GDP, uses GTPase motif to mediate binding of initiator met-TRNA to small ribosomal subunit

what happens when eIF2 is phosphorylated?

translation initiation is inhibited once eIF2B catalyzes exchange of GDP to GTP which activates eIF2 → turns into inhibitor of eIF2B which then blocks translation initiation

eIF2B

• limiting factor for initiation

• reactivates eIF2 by exchanging GDP for GTP for GTP to bind to eIF2 which becomes an inhibitor to block off translation

why does eIF2 phosphorylation shut down global translation?

due to eIF2B being limited and becomes a limiting factor for initiation

uORF (“upsream” open reading frame)

• starts at 5’ end of translation where context surrounding AUG can allow regulation → produce short polypeptides

• sequence starting with an AUG and ending with a stop codon → theoretically (not completely) able o encode a polypeptide in translation

• only gets “scanned through” in order for mRNA to remain bound → strategy to selectively increase a few proteins during stress conditions

how do uORFs or open reading frames regulate translation?

• ribosome starts at starting codon (AUG) and may not reach main coding sequence or main ORF

• some genes have short ORFs upstream of main coding sequence → if ribosome begins to translate a uORF it will terminate with the stop codon and fall off the mRNA before reaching to main codon sequence

ATF4 regulation

• translated more when eIF2 is phosphorylated → is transcription factor involved in responses to various stresses including amino acid starvation

• under non-stress conditions: translation of ATF4 is inhibited by uORFs

• under stress conditions: eIF2 is phosphorylated, reducing initiation at uORFs

IRES (internal ribosome entry site)

• allows ribosome binding without 5’ cap → allows ribosome to skip first AUG by binding to an internal site during translation

• allows two different protein sequences to be derived from single mRNA

• may sit between two separate ORFs, allowing independent simultaneous translation of two completely different proteins from one mRNA

• different initiation sites may lead to skipping of signal sequence (required for secreted/transmembrane proteins), and so switching between cytosolic and secreted forms of a protein

why do viruses use IRES?

host translation may be shut down

regulation of protein stability

• protein turnover → some proteins get rapidly degraded and then resynthesized when needed

• damaged or misfolded proteins must be destroyed to prevent accumulation of malfunctioning proteins

• ubiquitin (detect point of location to degrade or break down proteins)/proteasome system allows for regulated destruction of proteins → targeted protein is polyubiquitylated

  • proteasome recognizes polyubiquitylated protein and degrades it into short peptides

  • proteasomal degradation very energetically expensive

  • ATP required in need to unfold proteins for proteasome

epigenetics

heritable changes in gene expression without DNA sequence change of nucleotides → change in expression pattern but not nucleotide pattern or sequence

Epigenetic mechanisms

1. stable expression of a regulatory protein via a posiive feedback loop

2. covalent modification to histones, changing chromatin state

3. methylation of DNA on cytosine residues

4. stable changes in protein aggregation state (aka prions)

Positive feedback (epigenetics mechanism)

• once protein A made → maintains own expression and provides stable phenotype

• regulates it’s own synthesis until active signal leads to degradation

Histone modification (epigenetics mechanism)

• covalent modification of histones recruits enzymes that replicate same “histone code” when cell divides and maintains chromatin structure in daughter cells

• copies whatever histone code present in original strand onto newer chromatin from daughter cells

DNA methylation (epigenetics mechanism)

• methylation of cytosine in CG sequences suppresses gene transcription

• maintenance methyltransferase methylates CG sequences that are already paired with methylated CG → allow methylation pattern to be maintained during DNA replication

• will methylate on other side of strand

Protein aggregation state (epigenetics mechanism)

• some proteins can adopt alternate conformation that induces self-aggregation and also catalyzes conformation change in “normally” folded protein molecules

• from normal folded protein → misfolded protein or unstructured protein (prion) after conformation change