Gene Expression: Basic Mechanics of Transcription: Three critical phases 1. Initiation 2. Pause/Release 3. Elongation (Final)

What is transcription?

The process of making RNA from a DNA template

Which enzyme makes mRNA?

RNA pol II

What is a promoter?

A DNA region near the transcription start site where RNA pol II and general transcription factors assemble

What is an enhancer?

A regulatory DNA sequence that increases transcription of a target gene, sometimes from far away

Sequence specific transcription factors vs general transcription factors?

Sequence specific TFs bind particular DNA sequences at enhancers or promoters to target certain genes.

GTFs (TFIID, TFIIB, TFIIH) assemble at the promoter to position RNA pol II and start transcription

How can an enhancer regulate a distant promoter?

DNA looping brings the enhancer and promoter close together so their proteins can interact

What does cohesin do?

Helps organize and stabilize the DNA loop supporting enhancer promoter interactions

What does Mediator do?

A coactivator that connects the transcription factors to RNA pol II and the general transcription machinery

What is the preinitiation complex (PIC)?

RNA pol II plus the general transcription factors assembled at the promoter

Order of transcription initiation?

Enhancer loops to promoter, then sequence specific TFs bind enhancers, then Mediator links TFs to the machinery, then GTFs position RNA pol II at the promoter, then RNA pol II starts transcribing

How do transcription factors recruit the transcription machinery?

They bind DNA and recruit coactivators like Mediator, which summon GTFs that position RNA pol II and activate transcription

What would happen if a TF binds DNA but cannot recruit coactivators?

It recognizes the right DNA sequence but cannot pass the signal to Mediator, so the machinery is not recruited

What is promoter proximal pausing?

A temporary stop of RNA pol II about 20 to 60 nucleotides after it starts making RNA

Which proteins maintain pausing?

NELF and DSIF

Why is pausing useful?

RNA pol II is already started and positioned, so the gene can be turned on quickly (poised polymerase). It also adds a checkpoint and keeps chromatin open

What is transcription elongation?

RNA pol II leaves the pause and moves along the DNA making a longer RNA

What promotes release of paused RNA pol II?

P TEFb, which contains the kinase CDK9

What does CDK9 do?

Adds phosphate groups (phosphorylation) to proteins

What does P TEFb phosphorylate to release the pause?

NELF, DSIF, and the C terminal domain of RNA pol II

What happens to NELF and DSIF at pause release?

NELF is released and DSIF switches to supporting elongation

What are the three types of elongation factors?

Pausing factors (NELF, DSIF), pause release factors (P TEFb), and downstream elongation factors (Spt6, FACT, Paf1C)

What do downstream elongation factors do?

Help RNA pol II move through histone barriers in the gene body

What happens if P TEFb is inhibited?

Pause release stops and elongation of genes goes down

What is chromatin?

DNA packaged with proteins, including histones

What is a nucleosome?

The basic unit of chromatin: DNA wound around a histone core

How can nucleosomes interfere with transcription?

They can cover DNA sequences so TFs and other regulators cannot access them

What is chromatin remodeling?

Using ATP energy to change nucleosome position or structure so DNA is accessible

How can remodelers alter nucleosomes?

They slide, evict, or restructure them

Examples of chromatin remodeling complexes?

SWI/SNF (BAF), ISWI, CHD, INO80

Do remodelers add chemical marks to histones?

No, they only change nucleosome position or structure

A nucleosome blocks a TF binding site. What helps?

An ATP dependent chromatin remodeling complex that evicts or repositions the nucleosome

What are histone modifications?

Chemical groups added to or removed from histones that help regulate chromatin and gene expression

Five examples of histone modifications?

Acetylation, methylation, phosphorylation, ubiquitination, sumoylation

What are writers, erasers, and readers?

Writers add histone marks, erasers remove them, readers recognize them and recruit other regulatory proteins

Examples of histone writers?

HATs, histone methyltransferases (HMTs/KMTs), kinases

Examples of histone erasers?

HDACs and histone demethylases

What do bromodomains recognize?

Histone acetylation

What do chromodomains recognize?

Histone methylation

How does histone acetylation promote transcription?

It weakens DNA histone interactions, giving TFs and machinery access to the DNA

Does histone methylation always activate transcription?

No, it can activate or repress depending on which residue is methylated

A TF recruits a HAT. Effect?

Acetylation increases, chromatin opens, transcription is favored

A TF recruits an HDAC. Effect?

Acetylation decreases, chromatin is less favorable for transcription

Where is H3K27ac found?

Active enhancers and active promoters (strongly linked to active enhancers)

Where is H3K4me3 found?

Near active promoters and transcription start sites

Where is H3K79me2 found?

In transcribed gene bodies, often early to middle region (elongation mark)

Where is H3K36me3 found?

In actively transcribed gene bodies, increasing toward the 3′ end (elongation mark)

High H3K4me3 at a region means?

Likely an active promoter

High H3K27ac at a distal region means?

Possibly an active enhancer

H3K36me3 rising toward the 3′ end of a gene means?

Transcription elongation

What is mutational promoter analysis?

A method to find which promoter DNA sequences are important for gene expression

What is a transcriptional reporter assay?

A promoter is attached to a reporter gene (like CAT) whose expression measures promoter activity

How does mutational promoter analysis work?

Attach promoter to reporter, mutate or delete regions, then measure how reporter expression changes

What does a drop in reporter activity after mutation mean?

That region likely contains a regulatory element needed for transcription

What does no change in reporter activity mean?

That region may not be needed for promoter activity

Does promoter analysis identify which TF binds?

No, it only shows the mutated region is important

What is EMSA used for?

To test whether a protein binds a specific DNA sequence in vitro

How does EMSA work?

Mix labeled DNA with a protein sample and run a gel to see if a DNA protein complex forms

What is a shifted band in EMSA?

A band for a DNA protein complex that moves slower than free DNA

Why does bound DNA move slower in a gel?

The protein DNA complex is larger than free DNA, so it migrates more slowly

Does EMSA identify which protein binds?

No, more tests are needed to find which protein in the extract is binding

Limitation of EMSA?

It tests binding in a test tube and does not prove binding in living cells

What is ChIP used for?

To find whether a specific protein (like a TF) is associated with a DNA region in cells

What does ChIP seq do?

Identifies genomic regions bound by a protein

Four major steps of ChIP seq?

Crosslinking, chromatin fragmentation, immunoprecipitation, uncrosslink, and DNA sequencing

What happens in crosslinking during ChIP-Seq?

Formaldehyde links proteins to nearby DNA to preserve interactions

Why fragment chromatin in ChIP?

To break DNA into small pieces that can be isolated and analyzed so desired protein is exposed

What is immunoprecipitation in ChIP?

An antibody captures the protein of interest along with its bound DNA

What happens after immunoprecipitation in ChIP seq?

Crosslinks are reversed and the DNA is recovered and analyzed by PCR or sequencing

ChIP PCR vs ChIP seq?

ChIP-PCR checks specific, pre-guessed spots where TF was suspected to be, while ChIP-seq discovers every single spot across the entire genome where TF binds

What does a ChIP seq peak mean?

The region is enriched for the protein or histone mark being studied

What is CUT&RUN used for?

to identify the exact genomic regions where a target protein binds to DNA

How does CUT&RUN work, and how does it differ from ChIP seq?

An antibody binds the target and a tethered micrococcal nuclease (fused to protein A or A/G) cuts DNA right at the binding site. ChIP seq instead shears all chromatin and pulls down the complex

Why does CUT&RUN have lower background than ChIP seq?

Cutting is targeted near the protein of interest instead of fragmenting all chromatin

What is ATAC seq used for?

Finding accessible (open) chromatin regions across the genome

What enzyme does ATAC seq use?

Tn5 transposase, which cuts exposed DNA and inserts sequencing adapters (tagmentation)

What does an ATAC seq peak mean?

The chromatin is accessible, but this does not prove active transcription

What is ChRO seq used for?

Finding where RNA polymerases are actively making nascent RNA

How does ChRO seq work?

Isolate chromatin with engaged RNA pol, add biotin labeled nucleotides so polymerases extend their nascent RNA, then isolate and sequence the labeled RNA

ChRO seq vs ChIP seq?

ChIP seq shows where a protein sits on DNA. ChRO seq shows where active transcription is happening

Which technique to find where TF X binds the genome?

ChIP seq or CUT&RUN

Which technique to compare open chromatin between cell types?

ATAC seq

Why is the major groove important for TF binding?

It exposes chemical groups on the DNA bases so TFs can read the sequence without unwinding the helix

What chemical features are exposed in the major groove?

Hydrogen bond donors, hydrogen bond acceptors, and hydrophobic groups like methyl groups

How do TFs recognize specific DNA sequences?

By reading the pattern of chemical groups exposed by the bases, using alpha helices and loops that fit the major groove and amino acid side chains that make hydrogen bonds and other contacts on the DNA strand

Why do TFs make multiple DNA contacts?

Multiple contacts stabilize binding and add sequence specificity

Why does a TF's 3D structure matter?

It positions side chains to contact the correct DNA bases

What if a base in a TF binding site is mutated?

The chemical pattern changes and binding may weaken, strengthen, or be lost

Classic amino acid DNA base pairs?

Arginine forms two hydrogen bonds with guanine.

What four TF families were highlighted?

Homeodomain, zinc finger, glucocorticoid receptor, bZIP

What do all four TF types share?

DNA binding structures that let amino acids contact DNA to regulate transcription

What is a homeodomain?

A DNA binding domain found in TFs, especially developmental ones, with three alpha helices in a helix turn helix like structure

Roles of the homeodomain helices?

Helices 1 and 2 stabilize the structure. Helix 3 (recognition helix) enters the major groove and contacts DNA bases

What is a zinc finger?

A small DNA binding domain with two cysteines and two histidines held by a zinc ion (C2H2: a beta sheet plus an alpha helix that recognizes DNA)

Why do some TFs have many zinc fingers?

One finger recognizes about 3 bp, so multiple fingers read longer adjacent DNA sequences

What is the glucocorticoid receptor?

A steroid hormone receptor TF activated by glucocorticoids like cortisol. Its DNA binding domain uses two zinc coordinating modules to recognize specific response elements

What are the two regions of a bZIP TF?

The basic region (binds DNA) and the leucine zipper (promotes dimerization)

Why is the basic region called basic?

It has positively charged amino acids (lysine, arginine) that interact with DNA's negatively charged phosphate backbone

How does the leucine zipper work?

Hydrophobic interactions hold two subunits together in a coiled coil, positioning the basic regions on DNA

Why do TFs form dimers?

To increase binding stability, improve sequence recognition, and expand regulatory diversity

What holds TF subunits together?

Noncovalent interactions: hydrophobic, hydrogen bonds, electrostatic