BIMM 100 Final (COMEBACK BABYYY)

Genes in cell are alike but differ

since not all genes are expressed

Gene expression is regulated at multiple levels

Different cell types

Same genes → different mRNAs → different proteins → different morphology functions

Same cell type under different conditions

Same genes → different mRNAs → different proteins → different cellular behavior (Ex: +hormone or -hormone)

Gene organization in bacteria

One promoter regulates multiple genes (Operon - a cluster of genes under a single promoter)

During transcription will be transcribed into single mRNA (containing all genes)

During translation different parts of mRNA will be translated independently to independent proteins

How is pre-mRNA processed to mRNA in bacteria?

Recall: RNA Pol (ONLY one)

Does not undergo 5’ capping and 3’ poly A tail, instead ribosomes recognize site upstream of AUG (start codon) called ribosomal binding site (RBS) where Ribosome binds.

Operon consists of multiple genes how does Ribosome distinguish gene translation to produce separate proteins?

Find RBS at every individual gene found on Operon along with AUG (start codon) and UAA (any stop codon)

Sugar metabolism in bacteria

Consume glucose→glycolysis-energy ONLY until fully depleted, change gene expression (plateau until produce enzymes) to now digest Lactose ONLY

Lactose structure

Consists of Galactose-Glucose, Glucose→glycolysis→energy, and Galactose also digested/broken down, however need transporter lactose permease (brings Lactose into cell).

B-galactosidase used to break Lactose into Galactose-Glucose

Lac Operon

Lac operon promoter elements

CORE element (functional element) recognized by Sigma factor (general TF) that will recruit RNA Pol to bind to CORE element, upstream of CORE element find CAPsite (activating functional element) bound by CAP activator (will promote binding of Sigma factor to CORE element) promote transcription

Downstream of CORE promoter find Operator (inhibiting element) bound by lacI (trans acting) which will repress binding of Sigma factor to Core element

Find that a gene constantly produces both lacI and CAP activator

How is CAP activator regulated?

Glucose acts to inhibit CAP activator, Glucose reduces cAMP production → cAMP important for CAP-DNA binding

High glucose/ low cAMP & low CAP binding

Low glucose/ high cAMP & high CAP binding

How is lacI regulated?

Lactose acts to inhibit lacI, Lactose binds to lacI causing conformational change preventing binding of Operator

Operon expression in different conditions

Experimental method for studying Gene regulation

Western blot: To detect specific proteins and relative levels

Extract total proteins (no charge), mix with SDS + heat causing protein to denature, run Gel electrophoresis, transfer Gel on top of membrane to transfer, incubate membrane with proteins nucleic acid with antibody that bind to protein of interest (be able to see band)

Band intensity represents protein level

Eukaryotic Gene regulation

Whats the difference between Enhancers and PPEs

1.Can function over a long distance (found upstream, downstream, and inside introns), DNA is highly folded meaning the Enhancer could be found hundreds of nucleotides away but in reality is in proximity of CORE promoter

2.Position/Orientation of Enhancer does not matter still performs exact same function whereas position/orientation important for transcription direction in Promoter

Cis-regulatory elements

DNA seq that regulate gene transcription (anything found on DNA). Same in all cell types

Ex: Core promoter, PPEs, Enhancers, etc

How do cis-regulatory elements control gene expression?

Through TFs that bind to them

Trans regulatory elements

Transcription factors that bind to cis regulatory elements

Transcription factors are modular (functionally independent due to single protein containing multiple domains)

Find Activation/Repressor domain (regulates transcription) that is connected by Linker region to DNA binding domain (recognize specific DNA seq)

If remove Linker region and still have Activation/Repressor domain and DNA binding domain still observe transcriptional activity

Reacap Eukaryotic Gene regulation

All cells in an organism have the same DNA seq

But different genes are expressed depending on cell types , developmental stages, and environmental conditions

Controlled by cis regulatory elements and transcription factors bound to them

Cis regulatory elements: CORE promoters (general functional elements), PPEs and enhancers (both gene specific)

Trans regulatory elements

DNA binding domain components

Binding specificity - interaction with bases

Binding affinity - interactions with phosphate, sugars, or bases

How does activation domain promote transcription

TFII/Pol II recruitment to CORE promoter via mediator complex and/or activation domain itself

Chromatin regulation

Chromatin

DNA wrapped around histone (DNA more compact)

Function of Histones

protect dna and repress transcription

Histones are proteins (Proteins have N and C terminal)

Nucleosome

Repeated unit of chromatin, consists of 150bp DNA wrapped around 8 histones

Chromatin States (for a specific genomic region)

Heterochromatin (closed, inactive) - Chromatin modifiers allow transition between states but slow - Euchromatin (open, transcriptionally active) - Chromatin remodeling complex able to move position of nucleosome to free CORE promoter allowing recruitment of TFII/Pol II and FAST (w/ATP) - VERY ACTIVE Euchromatin state

Do all the genes have all three states?

No not all the genes have all three states

Chromatin modifiers and remodeling complex are recruited to specific DNA region by TFs or DNA binding proteins. Without unable to bind

DNase Protection Assay

To determine the chromatin state of specific DNA region, use restriction site and DNase (cut any naked DNA not gene specific) to cute specific region, Euchromatin each Nucleosome is cut but in Heterochromatin state unable since closed, remove histones and restriction site digestion → find multiple pieces of DNA in Euchromatin but one long piece of DNA heterochromatin, use radioactive probe, run Southern blot gel (run DNA on gel rather than RNA), will notice different length fragments

Chromatin modifications and modifer

Histone N-terminal contain many Lysine (+ charged), modifications are chemical marks added on specific lysine on histones

Modifier: Enzymes that add or remove these chemical marks

Chromatin modifications

Histone acetylation/deacetylation

Histone methylation/demetyhlation

Histone Acetylation/Deacetylation

Histone acetylation (loss of + charge, results in less tight binding in DNA, opposite charges attract) and bromodomains (transcription activators) bind to acetylated histones, promote Euchromatin state

Enzyme called HATs

Histone Deacetylation, enzyme called HDAC

Find that for each lysine N terminal there is a pair of modifiers for Acetylation or Deacetylation

Histone methylation/demetyhlation

No effect on charge of Lysine, instead promotes the Heterochromatin or Euchromatin state depending on Lysine. Methyl binds to protein with chromodomains (activators/repressors)

Modifers: Histone methyltransferase or Histone Demethylase

How do you get Heterochromatin formation and spreading

Often forms across a large genomic region, DNA binding elements bind to TF (repressor) recruit modifier (methyl) which will trigger modification of neighboring histones, once modified will bind to proteins with chromodomain, will further modify those neighboring histones forming cascade chain rxn (Heterochromatin spreading)

How is Heterochromatin spreading regulated/stop?

Find boundary elements (DNA sequence that stops Heterochromatin spreading)

How do boundary elements work?

Generate nucleosome free region (if no nucleosome neighboring will stop cascade)

Recruit proteins that will repress Chromatin modifiers

Chromatin Immunoprecipitation (ChIP)

To determine whether and where a specific protein binds to a specific DNA region (both are known) interaction in the cell (different cell types and environmental conditions yield different results)

1. Fixation (Formaldehyde) stabilize protein DNA interactions in cell

2. Isolate chromatins from cell, sonication (shear DNA into pieces, leaving only DNA that is protected by proteins/histones)

3. Immunoprecipitation: use antibody with bead (specific to TF, protein of interest) to pull down the protein and its associated DNA. Add to soln and antibody + bead + protein of interest will be left only

4. Reverse crosslinking: Release DNA from protein

5. Analyze DNA through PCR or Sequencing

Chromatin Immunoprecipitation (ChIP) can also be used to detect which chromatin region is modified

Same as previous experiment besides STEP 3 need antibodies that bind to histones (acetylated or methylated)

ChIP-seq use sequencing instead of PCR if interested in

areas where TF are bound/ modified(acetylated or methylated) across whole genome

Transcription regulation (activation)

Transcription activator (trans acting) its DBD bind specific region, Activation domain could recruit TFII/Pol II or chromatin modifiers (acetylation/methylation) or recruit Nucleosome remodeling complex (DNA helicase, ATP)

Transcription regulation (repressor)

Transcription repressor (trans acting) its DBD bind specific region, RD will either prevent binding of TFII/Pol II recruitment or repress Chromatin modifiers

Environment dependent gene regulation

Find Transcription factor (RD-DBD-LBD) is confined in the cytoplasm unable to reach target gene due to inhibitor and lack of presence of ligand. When ligand is present it dissociates inhibitor from Transcription factor and induces translocation of receptor to the nucleus to intiiate an anti-inflammatory response

Environment dependent gene regulation What happens once TF translocates into nucleus

Initiates anti-inflammatory response when binds to inflammatory genes, RD will recruit chromatin modifiers (Histone deacetylase, HDAC) therefore repressing affect

Tissue specific gene regulation

Pax6 is an important gene for the development of certain tissues, RECALL present in every cell, however only expressed in pancreas, eyes, and brain.

Brain specific TF/Retina specific TF/Pancreas specific TF bind to their specific tissue enhancer to activate expression, no other TF can bind to these enhancers

Gene regulation post-transcriptionally (SAME GENE)

Alternative splicing (only certain Exons present, addition/removal of Exon before transcription yields different protein), Alternative polyadenylation (STOP site present at different areas yielding different protein based on where STOP is recognized), Alternative promoters

Alternative splicing mechanisms

1. In pre-mRNA find short RNA seq called Splicing Silencers, recognized by proteins called Splicing repressor (repress recognition of splice sites)

Both Tissue 1 and Tissue 2 express splice silencers since are apart of gene, however splicing repressors only present in Tissue 2 since only Exon 1 and Exon 3 are expressed. Due to presence of Splicing repressor in Tissue 2 Exon 2 is recognized apart of Intron in between Exon 1 and Exon 3.

2. In pre-mRNA find short RNA seq called Splicing enhancers, recognized by proteins composed of RBD connected to SR domain (Ser-Arg rich domain, similar to AD). These proteins are called Splicing activators, you find weak splice sites in gene (w/o help Spliceosomes can’t recognize these sites and a result cut out Exon along with Intron), however with Splicing activators they activate these splice sites therefore allowing Spliceosome to recognize Exon and not cut off.

DNase cuts fragments unless it has a protein or Heterochromatin state protecting DNA, whereas restriction enzymes cut at exact seqeunce

TFII is a trancsription factor that contains TATA binding protein, it binds directly to

Promoters of open/active genes tp help recruit RNA Pol II

Linker scanning experiment

Mutation analysis, purposely mutate areas located in promoter. If mutation drops trancsription below wt (mutation ruins trancsription thus activator element)

If mutation increases above wtf (mutation causes over expression, therefore find a repressing element)

EMSA (Gel shift)

Measures whether protein binds to piece of labeled DNA

Key Differences Between DNA and RNA

• Structure: DNA is a double-stranded helix (like a twisted ladder). RNA is a single-stranded molecule, often shorter than DNA.

• Sugar Type: DNA contains deoxyribose (missing hydroxyl in 2’ instead two H) sugar, while RNA contains ribose sugar (which has one more hydroxyl group, making it more reactive).

• Location: DNA is found inside the nucleus and mitochondria. RNA is formed in the nucleus but moves to the cytoplasm and ribosomes.

• Stability: DNA is more stable, protecting the genome, whereas RNA is highly reactive and easily broken down.

Bases are

Purines are Adenine and Guanine(6 and 5 membraned ring) and Pyrimidines are Thymine and Cytosine (Only 6 membrane ring)

Triphosphate

Provides energy for polymerization

Nucleotide = Nucleoside Tri-Phosphate (NTP)

DNA: dATP, ddGTP, dCTP, dTTP (dNTPs)

What are the 5’ end, 3’ end composed of

5’ end has a free phosphate group (start of sequence) and the 3’ end has a free hydroxyl group (end of sequence or phosphate group connecting nucleotides)

Phosphodiester bond

3’ → 5’

Be able to interpret and conclude results of Genetic material is transferable experiment

Inject smooth to mice and dies

Inject rough to mice and lives

Inject smooth (heat killed) to mice and lives

Inject smooth(heat killed) and rough to mice and dies (this is due to transfer of genetic material that creates protein coat to evade human immune system)

Be able to interpret and conclude results of Genetic material using bacteriophage being DNA Hershey and Chase experiment

Through bacteriophages (S35 contained protein coat and P32 contained DNA), phages S35 (sulfur exists in protein not DNA) and P32 (only DNA has phosphorous not protein), saw that phages inject DNA into bacteria rather than protein, confirming DNA as genetic material

Base pairs

A=T and G=C in DNA but [A+T] ≠ G+C

G can form three H bonds with C

A can form two H bonds with T

DNA forms double helix, base found inside and phosphate outside, strands are antiparallel

Does the same rule of A=U and G=C apply in RNA?

No since RNA is generally single stranded

Chemical features of DNA

Highly stable

Negatively charged (phosphate groups)

Denaturing (melting) and annealing (H-bonds)

Is DNA replication conservative or semi-conservative, Meselson-Stahl experiment

Semiconservative: DNA helix split and each generates another strand

Conservative: DNA helix remains intact, but new synthesized DNA made

Found that DNA is semiconservative due to using parental strands synthesized in 15N and after replication introduce 14N, through Ultra-Centrifugation can separate strands due to weight.

Found that all newly synthesized DNA is 14N not 15N

What is required for DNA polymerization in vitro?

DNA synthesis is unidrectional

DNA polymerase, dNTPS, DNA single strand template and Primer (cannot start from scratch without these two)

DNA sequencing using chain terminators

ddNTPS (missing both OH groups unable to attack) cause visualization of multiple fragments, dNTP 3’ attacks phosphate of 5’ nucleotide

What role does the 3’ hydroxyl group play in addition to DNA polymerase

3’ OH group attacks alpha phosphate group and the diphosphate group leaves, underwent hydrolysis releasing energy, forming sugar bond

Energy: Triphosphate on nucleotides

Nucleic acid synthesis always 5’ to 3’ due to hydroxyl group attacking triphosphate, the 5’ end does not have hydroxyl group unable to attach new nucleotides

DNA synthesis in vivo (in cells)

DNA replication determined to be bi-directional in nature by treating with low radioactivity (thus nucleotides added appear) and after high radioactivity (thus newest nucleotides added are all highly radioactive) showed that nucleotides are being added both sides

What troubles are encountered due to bi-directional replication

Replication fork: Leading strand DNA synthesize and fork movement are parallel, however Lagging strand DNA synthesize and fork movement are antiparallel (requires multiple RNA primers). Nuclease removes multiple RNA primers along lagging strand. Different DNA polymerase synthesizes in gaps (however it can’t connect these DNA fragments), require Ligase (uses ATP) to connect phosphate sugar bond OH and Triphosphate

Each end has a leading/lagging strand

DNA polymerase functions

5’→3’ nucleic acid addition activity and 3’→5’ exonuclease activity (remove nucleotide if wrong)

Types of Nuclease

Endonuclease breaks phospho-sugar bond in middle of DNA (regenerate hydroxyl and phosphate) this is called a nick and is fixed by ligase

Exonuclease breaks phospho-sugar bond at end of DNA (3’ or 5’)

Ligase

Connect phospho-sugar bond between fragments on lagging strand

Gap in DNA described as

Missing nucleotides, this is created by endonuclease + exonuclease. Require DNA polymerase and Ligase to fill gap

Helicase

Breaks H bonds between base pairs

General strategy of repair of DNA single strand damage or replication error

1. DNA damage/error recognized

2. Nuclease removes nucleotides

3. Gap filled (DNA Pol + Ligase)

Promoter

Not apart of coding sequence rather recruit transcription factors and RNA polymerase

if no promoter → no gene product

5’ UTR (Untranslated region)

Occurs in nucleus as mRNA molecules is being syntehsized by RNA Pol II. Transcribed into mRNA

Coding region

Region that is transcribed into mRNA, only region translated into amino acid sequence to protein

3’ UTR

Occurs in nucleus as mRNA molecules is being syntehsized by RNA Pol II. Transcribed into mRNA

Molecular cloning: Transforming and obtaining bacterial clone library

Plasmid vector + dna of interest, insert dna into vector, mix plasmid with cell along with heat, cell will digest vector, plate w/amp, bacteria containing plasmid will only survive

Restriction enzyme

cut DNA at specific sequences (a specialized endonuclease)

Vector

Plasmid or viral DNA that can replicate in a desired organism

Each bacterial cell will take one single piece of DNA

What do we need in a plasmid/vector

Origin of replication (without wont be able to replicate

Selectable marker: antibiotic resistance gene (AmpR)

Gene of interest

Sticky ends

Generated by restriction enzyme, short single stranded DNA overhangs, can automatically form H bonds to complementary fragments (anneal: form H bonds)

During transformation of plasmid and bacteria, one bacterial cell can only take up one plasmid, seperated by applying amp and only those with ampR gene survive and produce colonies

If there is no origin of replication even if has ampR gene, cell will not recognize plasmid, and no replication of DNA. Will not see any colonies (only few amounts) under Amp

Gel electrophoresis to separate DNA molecules based on size

Molecules move through pores in gel (agrose), DNA is negatively charged so goes from - → +. Smaller fragments able to travel faster rate than larger fragments

Genome sequencing

Genomic DNA, use a restriction enzyme to cut specific sequences of DNA, from there you ligate the fragments with vectors (w/ origin of replication and ampR gene), put on dish and watch colonies grow (put amp) to kill off any colonies not resistant to amp and divide into separate test tubes, from there you align and get whole genome seq. Are able to determine order by sequencing genome again but with different restriction enzymes and if see overlap are able to confirm fragment order

Steps of PCR

Require two primers (since double stranded DNA)

Denature DNA by heating up to 95*C (single strand)

Annealing of primers by cooling 50-72*C

Elongation/Extension of primers (polymerase + dNTPs makes new DNA) 72*C

Design Primers

Forward and Reverse and are just complementary sequences

Single digest (one singular enzyme, found on plasmid)

Opens plasmid into one long piece (only see one band)

Double digest (multiple enzymes same/different)

distance between two enzyme sites