HLSC322 exam 4 - can't believe there's more!

Polycistronic vs Monocistronic mRNA

encoding several proteins from same gene vs. encoding only one protein from one gene, occurs in prokaryotes and eukaryotes respectively

Cis-Regulatory Element (CRE) vs. Trans-Regulatory Element (TRE)

both regulate transcription, though former is noncoding DNA sequence located on the same chromosome as the promoter, while latter is diffusible molecule encoded by gene on different chromosome 

Levels of DNA condensation

Nucleosome, 30 nm fiber, 300 nm loops, 250 nm fiber, chromatid

Histone modifications

Histone methylation may either open or close chromatin / activate or repress transcription depending on location. Performed by histone methyltransferases

Histone acetylation generally opens chromatin and activates transcription (reduces + charge on histone tails, weakening affinity for DNA). Performed by histone acetyltransferases

Modifications are passed on to daughter cells ft. maintenance methylases / acetylases

Topologically Associating Domains (TADs)

foundational 3D structural units of chromatin, typically 200 kb to 5 Mb in size, where DNA interacts frequently within themselves but rarely with outside regions (important for gene regulation, i.e. correct enhancer-promoter interaction)

Chromatin remodeling proteins

multi-subunit protein complexes that expose DNA at promoter / CRE by removing / repositioning nucleosomes

Pioneer factors

specialized transcription factors that can bind to closed chromatin / nucleosomal DNA and induce local decompaction so other factors can begin assembling at promoter

Coregulators

proteins that interact with transcription factors to activate (coactivators) or repress (corepressors) gene expression by remodeling chromatin.

• Histone methyltransferases 

• Histone demethylases 

• Histone acetyltransferases (HATs) 

• Histone deacetylases (HDACs) 

ChIP-seq

Chromatin Immunoprecipitation sequencing, a technique to determine which DNA sequences are bound to a certain protein

Cross-link DNA and bound proteins, incubate in antibody specific to protein of interest, pull proteins of interest out, separate out DNA and sequence

Gel shift assay

anotha technique to define DNA-binding sites on a protein.

A DNA sequence (probe) thought to contain the binding site is labeled (eg. with radioactive phosphorus isotope ³²P)

Running the gel:

Lane 1: probe only

^{check for purity of DNA and obtain baseline migration speed to compare to protein-bound complex}

Lane 2: probe + protein suspected to bind

^{if binds, should migrate slower than 1 = “shift”}

Lane 3: probe + protein + competing DNA

^{should see no shifted band, indicating competitor used up protein binding sites → binding site was correct}

Lane 4: probe + protein + noncompeting DNA

^{check that binding is actually at particular sequence and not just binding to any random DNA}

Lane 5: probe + protein + antibody

^{confirm identity of protein. If binds, should migrate slowest = “supershift”}

Reporter gene

molecular tool used to study gene regulation by encoding easily detectable proteins—eg. fluorescent proteins (GFP), enzymes (lacZ/𝛽-galactosidase), or luminescent proteins (luciferase)—that produce measurable signals.

By attaching these genes to a target promoter, researchers can measure promoter activity, visualize protein localization, or monitor cellular signaling pathways in real time

Basal transcription factors

bind to core promoter (tho not necessarily directly to DNA itself) and recruit RNA polymerase

TATA box

conserved DNA sequence (usually TATAAA) in eukaryotes, usually 25-35 bp upstream of transcription start site. Binding site for TATA-binding protein (TBP) which recruits other TFs and eventually RNAP 

Insulators

sequences between enhancer and promoter that prevent the 2 from interacting, often involving CTCF binding and formation of chromatin loops as physical barriers

CCCTC-binding Factor (CTCF)

binds DNA ft. zinc fingers

• establishes TADs

• binds insulator sequences → DNA loops wherein enhancers can only activate promoters in same loop

Jun & Fos

Transcription factors important for cell growth, proliferation and differentiation (expressed more highly in response to growth factors and/or stress). 

Jun-Fos heterodimers are more stable / have higher DNA binding affinity than Jun-Jun homodimers, so they are more common in most cells. 

Jun-Fos bends DNA towards major groove while Jun-Jun bends towards minor groove.

Myc & Max

Transcription factors with the same DNA-binding sequence and dimerization domain, but only Myc contains trans-activation domain 

Myc-Max heterodimers have higher binding affinity than Max-Max homodimers 

Myc will displace one of the Max and form the heterodimer → activate cell proliferation genes