MCB 110 MT 2

Promoter

Enhancer/Silencers

• Where transcription starts

• Adjusts transcription level

Coding/Template Strand

RNA sequence matches coding strand, but is transcribed from template strand, 5’ to 3’ direction

RNA secondary structure and tertiary structure

Secondary structure: ssRNA, antiparallel helix(hairpin, stemloop)

Tertiary structure: ssRNA forms non-base-pairing interactions, stabilized by positive metal ions

RNA composition

Mostly rRNA (ribosomal), then tRNA, mRNA

Most RNA is not mRNA

RNA synthesis

Synthesized from NTPs, incorporated with PPi release, and the first alpha phosphate from the NTP is incorporated into the RNA

Transcription Initiation/Elogation Rates

Bacteria: more initiation, slow transcription rate; short transcripts, takes a few minutes

Eukaryotes: less initiation, fast transcription rate (bursts); long transcripts, takes hours

Gene expression regulation

Degradation and synthesis are both regulated, depends on abundance of the mRNA, ratio of synthesis and degradation, and life cycle phase.

Ex. histone genes are only expressed during S phase, bacteria regulates metabolic enzyeme genes depending on what needs nutrients avaliable, etc

RNA gel electrophoresis

RNA denatured to eliminate secondary structure, only rRNA and tRNA visible

Northern Blotting

Separate RNA by gel electrophoresis, then transfer RNA from gel to membrane, hydridize probe with complementary sequence

Used to measure promoter regulation under different conditions by analyzing RNA length

RT-qPCR

Reverse transcriptase turn mRNA template into cDNA through random priming, cDNA is then PCR amplified; results show up as exponential curves, where 2x more template = 2 fewer cycle

RNA-seq

RNA is extracted from cells, undergoes reverse transcription, then used for Illumina high-throughput sequencing; results show up as count, where more sequences = more cDNA = more mRNA

Bacterial gene organization

Begins from promoter, ends at terminator (multiple possible), operon contains multiple genes (unidirectional)

Bacterial RNA Pol

Made up of 5 polypeptides, no proofreading exonuclease, cannot recognize or open a promoter (requires sigma factor)

Coordinates NTP incorporation, strand separation, and RNA produced

Sigma Factor

Binds to promoters (TTGACA, TATAAT) in 2 positions (major groove of alpha helix) and contacts RNA pol

Closer match to consensus = higher affinity = more RNA transcription

Separates RNA strands at promoter into open complex

number of sigma factors > number of pol —> sigma factor competition

Alternate sigma factor mechanism (flagellar biosynthesis)

FlgM blocks sigma F factor, but when FlgM is pumped out of the cell through the flagellum, sigma F becomes active and transcribes to make more flagellum

Abortive Initiation

After RNA strand starts and proceeds 8-15 nucleotides, RNA is released to clear promoter sequence; sigma factor released as a result and elongation starts

RNA Pol Stalling/Transcript Cleavage

RNA pol can stall (wrong nucleotide, protein in the way, etc) and backtrack several nucleotides to fix this

Transcript cleavage factor GreB cleaves mRNA after backtracking and allows new elongation

Elongation factor NusA helps to prevent stalling

Intrinsic/Rho-independent Termination

Stable RNA hairpin forms, and RNA falls off, transcription ends (no proteins needed)

Rho-dependent Termination

Termination factor Rho binds RNA at rho utilizaiton site as a hexamer ring, threads RNA through the pore using ATP hydrolysis, destabilizes RNA and ends transcription

Synthetic RNA Production

T7 RNA pol used: derived from T7 virus, promoter-binding protein built in, highly active

Used to make specific proteins, synthetic RNA probes and mRNA vaccines

Bacteria Metabolic Genes

trp operon: tryptophan biosynthesis

• repressor requires tryptophan to bind DNA and block transcription (allosteric change in repressor)

lac operon: lactose utilization

• Lac repressor LacI blocks promoter, and is released when lactose binds (strongly on in low glucose, high lactose; weakly on in high glucose, high lactose; off otherwise)

Genetic analysis of regulatory genes

Mutant lacking repressor: genes are always transcribed (no tryptophan/always glucose)

Mutant lacking activator: no transcription

Mutant at DNA binding site: loss of protein, affects one operon

Electrophoretic Mobility Shift

Gel shift assay: measures protein-DNA complex formation since it moves more slowly (ex. TtgR regulator with Cml/Tet)

Properties of Regulatory Proteins

Makes base-specific contacts with major groove (high specificity and affinity)

Many bind palindromic sites as dimer (lambda repressor)

Nuclease Footprinting

Used to map protein binding sites; protein binding prevents nuclease activity, DNase added to DNA, and uncut regions = footprint

CAP + Cyclic AMP

CAP: catabolite activator protein; binds alpha in RNA pol and recruits it to promoter, can favor both open and closed formations

Cyclic AMP: small molecule signal for low glucose; intermediate between ATP and AMP

• CAP:cAMP activates transcription for other alternate carbon sources (ex. lactose)

• cAMP rearranges CAP by rotating helix to align with major groove

Sensor Kinases

Sensor kinase: PhoR

• activated by low phosphate, autophosphorylates itself on histidine, transfers phosphate to aspartate on PhoB

Response regulator: PhoB

• activates downstream responses

Anti-Termination

Bacteriophage lambda infection: inserts terminators tL and tR into genome, makes lambda N protein which prevents termination by interacting with elongation factors NusA and NusG (blocks Rho)

RNA binding (small molecule + riboswitches)

Small molecule: TPP binds to RNA as a ligand; high affinity and specificity

Riboswitches: metabolite binds to aptamer domain, causes structure changes to effector domain; can affect intrinsic terminator

• If metabolite (ex. nucleotides, amino acids, ions) present/absent, transcription can be switched on/off

Eukaryotic gene organization

Different RNA pols for different functions, RNA Pol II transcribes mRNA

mRNA is processed co-transcriptionally: 5’ cap addition, splicing to remove introns, Poly A tail addition

Eukaryotic RNA Pol II

Homologous to bacterial RNA Pol with 5 polypeptides, but has 7 extra proteins

Contains CTD: C terminal domain

• Many repeated copies of YSPTSPS: chemical modified during transcription to regulate different gene expression

Also stalls and backtracks like bacterial RNA Pol, elongation factor TFIIS cleaves transcript like bacterial GreB

General transcription factors

TFIID: consists of TBP plus other proteins

TBP: TATA-binding protein; binds to TATA box

TFIIB: single protein that binds to BRE sequence

These transcription factors bind to promoter before RNA pol arrives

TFIIH

Eukaryotic equivalent of sigma factor: drives open complex formation

Contains XPB which is a ATP-dependent helicase, opens DNA

Mediator Complexes

Mediator complexes helps to connect regulatory TFs to Pol II machinery; can modify histones or remodel nucleosomes

ChIP-Seq

ChIP: chromatin immunoprecipitation

• chemically links protein to DNA, DNA is cut, protein (RNA Pol II) captured with antibody, antibody removed, DNA purified to sequence

ChIP-Seq:

• High-throughput DNA sequencing, counts DNA fragments and pieces them together, compared to control DNA is identify where RNA Pol was bind to

Nucleosomes

Barrier for transcription since DNA is wrapped around histones

Elongation factors (DSIF) assist polymerase to transcribe without stopping

Eukaryotic Transcription Termination

CTD recognizes 3’-end processing signals on mRNA and cleaves mRNA product; downstream RNA is degraded by XRN2 nuclease

CTD Phosphorylation Cycle (General Steps)

Free polymerase, pre-initiation (Ser5 phosphorylated), initiation, elongation (Ser2 phosphorylation), termination (phosphorylation removed)

CTD Initiation

Ser5 phosphorylated by major kinase Cdk7 in TFIIH

ChiP seq shows high density right after the promoter (indicating pausing of Pol II near promoter)

Promoter-Proximal Pausing

Poll II starts transcription and moves 20-60 bases, then NELF and DSIF are recruited by Ser5-P to cause pausing; pausing is a regulatory checkpoint

Elongation factor P-TEFb relieves pausing by phosphorylating DSIF and Ser2

DSIF

Elongation factor; contains Spt4 and Spt5, where Spt5 is homologous to bacterial NusG; when phosphorylated, it promotes transcription elongation

Kinases and Phosphatases for CTD

Cdk7 in TFIIH for Ser5-P

Cdk9 in P-TEFb for Ser2-P

Ssu72 phosphatase to remove Ser5-P

Nuclear Run-On Transcription (GRO-seq)

Isolates nuclei (RNA Pol II remains on DNA), add NTPs and lebeled NTPs, allows transcription to continue and labeled nucleotides incorporated, purify and sequence to identify active polymerases

• Ex. Bromo-UTP: bromouridine = modified uracil base with bromine; can be pulled down with antibody to isolate RNA

ChIP for RNA Pol II Phosphorylation

Crosslink DNA with RNA Pol II, fragment and pulldown with Ser5-P and Ser2-P antibodies, sequence to compare DNA where different states of Pol II can be found

• Ser5-P will be enriched near promoter, while Ser2-P will be enriched in gene body

Processing Enzymes

1. Capping: happens near start of transcription, capping enzyme Ctg1 reads more than one Ser5-P using lysine and arginine which forms salt bridges with phosphates

2. PolyA tail + cleavage: happens near end, cleavage enzyme reads Ser2-P

Phosphorylation makes a protein very negative and large, easy to recognize

Enhancers

Eukaryotic regulatory sequences, can be very far from promoter; autonomous promoter = core promoter + nearby enhancer

Although far in sequence, they are not far physically because DNA loops around, proteins at the enhancer still interact with proteins at the promoter

Co-activators + Co-repressors

Co-activator: increases transcription by binding to TFs

Co-repressor: vice versa

Small number of co-activators and co-repressors, shared by many TFs

TFs can bind at distant enhancers and regulate a core promoter far away on DNA

Reporter Assays

Test enhancer: put candidate DNA upstream of core promoter and reporter gene

Test promoter: put enhancer upstream

TF expressed from plasmid, reporter gene with binding site (LacZ, luc, GFP)

Mapping Regulatory Sequences

Take a control region and make mutations/deletions, measure transcription to see which mutation increases/decreases transcription

Zinc Fingers

DNA binding domains in eukaryotic TFs, each finger recognizes 3-4 base pairs, Zn 2+ coordinated by cysteine and histidine

Other examples of gene control

1. Dimerization of TFs can bind different sequences

2. NFAT and API form strong cooperative binding in each other’s presence

3. Enhanceosome (interferon-beta promoter): large protein complexes assembled on DNA, controls immune response gene (interferon)

Activation Domains

Disordered when alone, but folds when binding partner

CREB (human cAMP activator protein): binds CBP co-activator

Gal4 activation domain: Gal4 clocked by Gal80 in off state, galactose binds Gal3 which binds Gal80, releasing Gal4; Gal4 activation domain recruits mediator

ELK1 activator protein: bound to DNA but inactive in off state, mitogen growth factors cause ELK1 phosphorylation, allows it to interact with co-activators and mediator

Mediators

Large complex of 25 proteins, bridge between activators and RNA Pol II; activators do not directly bind RNA Pol II

Assembles with activators at enhancer before RNA Pol II, then mediator bind GTFs and RNA Pol II CTD (non-phosphorylated); mediator then recruits TFIIH to phosphorylate Ser5 on CTD

Drosophila + HIV Tat Protein

Drosophila: heat shock response; HSF (heat shock factor) TF binds DNA and releases paused polymerase, recruits P-TEFb, transcription continues

HIV Tat Protein: viral RNA has TAR hairpin, Tat binds TAR and recruits P-TEFb, releases paused polymerase, transcription continues