Molecular Biology (BIO 99)

UCI, Tsai, second half of quarter

Operon

Operon: A group of genes that are transcribed together primarily in prokaryotes under the control of a single promoter. It includes an operator, promoter, and structural genes.

AT MINIMUM IT REQUIRES A GENE CLUSTER AND PROMOTER

Promoter sequences in E. Coli

-35 box, -10 box, and purine start sites are most common in E. Coli

-35 box is the major site for sigma 70 (E. Coli sigma factor)

-10 box/pribnow box → important factors

AT rich → easier to unwind due to double bonds

major binding site

In DNA footprinting, on a gel electrophoresis with specific binding proteins targeting for the promoter → what does the empty space represent on the electrophoresis?

where the promoter sequences are

RNA polymerase → significant physiological aspects

3’ -OH attacks the 5’ triphosphate through a nucleophilic attack

there are 2 Mg2+ cofactors

!!THERE IS NO NEED FOR AN RNA PRIMER (LIKE IN DNA POLY)!!

RNA polymerase subunits & their functions

Alpha → Function: Essential for assembly and elongation

Beta →Function: Binds NTPs and interacts with sigma subunit → polymerizes RNA (!!SUBUNIT WITH POLYMERASE ACTIVITY!!)

Beta' → Function: DNA binding

Sigma → Function: Recognizes promoter sequences.

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Omega (not as important) → functionality and stability

Subunits of the core enzyme?

(alpha)₂(beta)(beta’)

note that the 2 denotes there are 2 alpha subunits

core enzyme + sigma cofactor

holoenzyme

what model indicates how DNA is pulled into RNA polymerase?

The “scrunching” model

Rifampicin binds to what and does what?

binds to? → BETA subunit

does what? →BLOCKS INITIATION

What has the highest speed among the 3?

DNA replication — RNA transcription — Protein translation

DNA replication

Direction of transcription is 5’-3’ → When translating the 3’ end is correlated to ________ supercoils(________) and 5’ end is correlated to ________ supercoils(________)

Direction of transcription is 5’-3’ → 3’ end is correlated to positive supercoils(unwinding) and 5’ end is correlated to negative supercoils(rewinding)

What helps rescue an arrested/stalled complex?

GreB (transcription factor)

Rho-independent termination of transcription involves?

GC-rich inverted repeat (forms stem loop) and U-rich stretch (causes pausing and release)

Rho-Dependent termination of transcription involves what?

Hexameric (!!6 RHO FACTORS!!) ATP dependent helicase

Transcription VS DNA replication

Similarities

DNA is a template

Synthesis of Complementary strand

Same mechanism of phosphodiester bond formation

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Differences

Transcription is selective

1 strand is used as a template for transcription

!!!!Transcription does not require a primer!!!! (most important)

Transcription is more error prone (no exonuclease activity)

Why regulate gene expression?

Environmental factors: food sources change

Developmental/Differentiation

Cell specialization

What is the most common method of regulation in gene expression?

Controlling transcription initiation is most common → most efficient to regulate at beginning of pathway

Negative control VS Positive control

Negative control inhibits transcription (repressor)

Positive control activates transcription (activator)

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DOES NOT MATTER IF A REPRESSOR IS RELEASED FROM A SILENCER AND “ACTIVATES TRANSCRIPTION” OR IF A ACTIVATOR RELEASES FROM A PROMOTER AND “INHIBITS TRANSCRIPTION” THE MODE OF REGULATION IS WHAT DETERMINES THE TYPE OF CONTROL

Lac Operon major products and their functions

LacZ → Lactose degraded by beta-galactosidase (Beta-gal)

[No LacZ = No transcription bc no side product of allolactose]

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LacY → Galactoside permease for uptake of lactose

[No LacY = No transcription bc no lactose can enter the cell]

What modes of regulation does the Lac operon contain?

Both positive and negative regulators

Lac operon “

Promoter I (P_I)

LacI: regulatory

PromoterL (P_L)

OperatorL: regulator binding site (O_L)

LacZ: B-gal

LacY: permease-transport

LacA: galactoside transacetylase

Concept: If LacY is knocked out in one strain of bacteria and LacZ is knocked out in another strain of bacteria and they are cultured individually with a media of [Lactose+/Glucose-] what will the net effect be?

both strains will exhibit no growth

Concept: If LacY is knocked out in one strain of bacteria and LacZ is knocked out in another strain of bacteria and they are cultured together with a media of [Lactose+/Glucose-] what will the net effect be?

If the knockouts are combined then there will be growth because of the diffusible protein products

What is the ONLY condition in which the Lac Operon will be active?

NO glucose-

AVAILABLE lactose+

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ANY other condition will facilitate the use of glucose catabolism OR no catabolism whatsoever

Given conditions glucose+ and lactose- what will be the effect on the Lac Operon?

No Lactose and w/ Glucose:

repressor (I) active, binds operator, inhibits RNAP to make Z,Y,A

Allolactose

side product of Beta-gal, and an inducer (IPTG is also an inducer) (de-represses the repressor)

what are the 3 operators of the Lac Operon

-82 (O3) and +412 (O2) are auxiliary, +11 is main operator (O1)

Lac repression binds what and needs how many proteins?

4 Lac_I products is necessary to inhibit → dimerization at O1, and dimerization at O2/O3 → Dimers form another dimer → DNA loop

What sequences do the lac operator have?

Why is this significant?

palindrome → Lac_I is a dimeric protein and palindromic sequences are recognized by dimeric proteins

Lac repression involves DNA looping

Cis vs Trans gene regulation

Cis acting elements → DNA sequences in vicinity of genes

Trans acting factors → diffusible protein factors that bind to DNA sequences

Regulatory mutants

i- → mutant nonfunctional repressor protein

I^S → super repressor: cannot be inactivated by inducer

O^C → operator constitutive: cannot be bound by repressor

p- → nonfunctional promoter

CAP protein

High glucose levels outside cell create low cAMP levels within cell

!!!CAP/cAMP = gene activator!!! MOST IMPORTANT

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CAP = CRP = “cAMP receptor protein” → Bind DNA when activated by cAMP → Binds lac operon promoter for lacZYA

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[NO CAP/CRP ACTIVATION = NO TRANSCRIPTION]

Catabolic vs Anabolic operons

Lac operon encodes catabolic enzymes:

Catabolic operons generally regulated through induction

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Trp operon encodes anabolic enzymes:

Anabolic operons generally regulated through repression

Trp Operon: 2 modes of regulation and their nuances

Trp repression:

trpR requires trp as a corepressor → tryptophan levels low → no corepressor → repressor does not bind to aporepressor → no binding to operator → no repression → Expression of trp operon

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Transcription attenuation:

High trp → leader sequence made (trpL) exclusively

[note there is 2 critical Trp codons in the leader sequence that are sensitive to Trp levels in the cell → if these sequences are altered and changed to different AAs the sequence will respond to the new AA]

LEADS TO ALTERNATIVE BASE PAIRING:

1. If 3-4 pair: structure forms → attenuator → acts as a transcription terminator

2. if 1-2 pair it leads to 3-4 pairing and it ALSO leads to transcription termination

3. if 2-3 pair it becomes an ANTI-terminator and PREVENTS 3-4 from binding

Trp Operon: Operon Order

trpP (promoter)

trpO (operator)

trpL (leader)

trpE

trpD

trpC

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first 3 are the MOST imporant

Prokaryotic DNA vs Eukaryotic DNA:

Information density: prokaryotes greater than eukaryotes

Eukaryotes have repeating DNAs → prokaryotes have unique DNA

Genome association w/ protein (Chromatin)

what percentage of the human genome encodes for proteins?

1% of human genome encodes for proteins

Renaturation kinetics’/Reassociation kinetics’ significance

When viewing a graph, Prokaryotes have 1 jump in transition and Eukaryotes may have multiple jumps

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this is because prokaryotes have ONLY unique DNA while Eukaryotes have multiple classes of DNA ranging from extremely repetitive sequences (telomeric DNA for example) to extremely unique sequences (coding DNA)

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[“3 classes” is considered regular for eukaryotes

“1 class” is considered regular for prokaryotes]

number of of RNA polys dependent on domain (Eukaryote vs Prokaryote)

3 eukaryotic RNA polymerases vs 1 prokaryotic RNA polymerase

What does eukaryotic RNAP require a lot of?

multiple transcription factors

[Note that we have roughly 3000 TFs → these bind together to encode our genes which number roughly 25000]

[Note that combinations of these TFs may activate different genes as well]

What does post-transcriptional processing involve?

splicing, capping, polyadenylation of mRNA

What is the point of modification of chromatin/looped domains?

to access genes

[chromatin and looped domains exist to condense DNA → therefore modification of them exists to access the inaccessible genes and modification to return them to their previously compacted state]

Eukaryotic core promoter motifs

TATA box, Initiator element (Inr), DPE, TFII recognition element (BRE)

Preinitiation complex formation

TFIID→TFIIA→TFIIB→TFIIF-Pol(joins together)→TFIIE→TFIIH

DAB → F-Pol → EH

these are called the “general transcription factors:

TFII = “ Transcription Factor ” — RNA Polymerase “ II ”

TFIID important aspects

Made up of TBP(TATA-binding protein) and TAFs (TBP-associated factors)

Binds DNA in the minor groove

TFIIH important aspects

DNA helicase activity/ATPase activity

Kinase activity

Mutations in TFIIH potentially lead to what 2 diseases?

Xeroderma pigmentosum

Cockayne syndrome

CTD of large subunit of RNAP helps transition from initiation to elongation → what important aspects of the CTD help facilitate this and what needs to happen to the CTD to begin elongation?

CTD contains many tandem repeats of the heptapeptide Tyr-Ser-Pro-Thr-Ser-Pro-Ser

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TFIIH has to phosphorylate RNA pol II CTDs for RNA elongation to occur

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Ser 2 and Ser 5 are important →

• RNA pol II is phosphorylated at ser5 to initiate elongation

• RNA pol II is phosphorylated at ser2 after bp +50 during elongation

• CTD must be phosphorylated on Ser-5 (negatively charged attracts positive enzyme—attracts capping enzyme to make 5’ cap)

what 2 proteins regulate elongation?

NELF → Negative elongation factor

DSIF → DRB-Sensitivity-Inducing-Factor

Transcription Elongation factor: Fork Loop 1

prevents premature unwinding

Transcription Elongation factor: Rudder

prevents the DNA to rebind to mRNA

Transcription Elongation factor: Lid

wedge and guide the incoming DNA

Transcription Elongation factor: Bridge Helix

acts as a ratchet (circular motion in one way)

mRNA processing (3 steps)

5’ Cap needs to be added

Splicing the mRNA

Cleavage/Addition of the Poly-A tail

5’ Cap (enzymes/important factors involved in creation)

5’-triphosphate Step 1

Guanylyltransferase Step 2

N7G-methyltransferase Step 3

Alternative Splicing errors may lead to?

Errors may lead to muscular dystrophy

4 elements for splicing

GU-rich sequence is the 5’ splice site

AG sequence 3’ splice site

Branching nucleotide → Adenine

Pyrimidine rich tract

Splicing steps

1. 2’-OH of adenine is the nucleophile (!!! IMPORTANT TO KNOW THAT NUCLEOPHILE IS THE 2’-OH OF ADENINE !!!)

2. The Branching Nucleotide’s 2’-OH attacks the 5’ GU

3. 3’-OH of the 5’ GU that was removed attacks the AG at the 3’ splice site

4. Exon is separated from intron

Functions of poly(A) tail

Protects from exonuclease activity

Important for transport of mRNA

Important for translation

Allows for isolation of mRNA in a lab

Antitermination model for termination vs Torpedo model of termination

Which of the 2 is right and what does it propose?

Torpedo Model is correct.

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RNAP is stalling and slowing down after making polyA tail → RAT1/Xm2 attacks the poly(A) tail → ends the termination after degrading rest of mRNA (torpedo model) (!!! enzymes involved are important !!!)

mRNA export and import requires what?

GTP hydrolysis

1. mRNA out needs Exportin and Ran + 1 GTP

2. mRNA in needs importin and Ran + 1 GTP

Euchromatin vs Heterochromatin

Euchromatin → loosely packed DNA (more expressed)

Heterochromatin → tightly packed chromatin

DNA binding and activation/repression domains:

Transcription factors have a bimodal composition:

One domain recognizes a specific DNA motif or “DNA binding domain” and the other interacts with proteins

DNA binding motifs

Zinc-finger sequence motif

• Binds to major groove

• [has little to no constraint to bind inverted repeats as it does not form dimers]

• [if Tsai brings up “Frankenstein nuclease” or smth immediately think Zinc-finger]

Helix-turn-helix motif

• Binds to major groove

• Form dimers [note that if one of the proteins is mutated it will not function properly or if it is introduced as the mutated protein it will cause the wt gene to not function properly]

• [9 proteins need to be bound]

Leucine Zipper

• Leucines are spaced 7 proteins apart

• Form dimers

Eukaryotic promoters are sequences bound by what

Sequences bound by PIC (Core promoter)

transcriptional activators (regulatory promoters)

how far can an enhancer be from a promoter? Where can it be? What does it cause?

Separation from enhancer may be in several kbps to the promoter VERY FAR

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Enhancers can be upstream or downstream and still provide the same function

Activators VS Repressors

Activators(protein/trans factor) bind to enhancer sequences(DNA/cis factor)

Repressors(protein/trans factor) bind to silencer sequences(DNA/Cis factor)

Repressors interfere with the functioning of activators and slows transcription, what are the modes of repression?

May either interfere with the binding site of the activator (the enhancer sequence) (competitive DNA binding)

may bind to the activator as it may “mask the activation surface” of the PIC

May bind to the GTFs in the PIC and “directly interfere with the binding of the activator” on an enhancer sequence to the PIC

What are the various things that the CTD attracts?

Termination factors, splicing factors, elongation factors, and mediators, [also attracts the capping enzyme when ser2 and ser5 are phosphorylated by TFIIH on the heptapeptide repeats]

CTD acts as an assembly line for tools needed for promoter clearance and RNA processing which is coupled to the elongation process

mRNA might get fed through this line of factors which are bound to the CTD

Insulators

block activation by enhancers, or block repression by silencers.

Histones are made up of how many proteins?

octamer (2xH2A,H2B,H3,H4) — 8 proteins to make up the histone

[Note when the histones are modified the H3 and H4 pairs move as a unit, and the H2A and H2B pairs move as a unit as well]

How many base pairs wrap around a histone and how many turns?

146 bps wrap around a histone (2 turns)

What types of AAs do histones mainly contain?

Histones contain many basic amino acids (Lysine & Arginine)

positive charge attracts negative DNA

How to alter DNA packaging

Chromatin Remodeling

Histone tail modifications

Histone tail modifications: Acetylation

HATs (histone acetyltransferase)

Enhances transcription

[can change the net charge on the histone’s surface]

Histone tail modifications: Deacetylation

HDACs (histone deacetylases)

Represses transcription

[can change the net charge on the histone’s surface]

Histone tail modifications: Methylation

Leads to repression

Histone tail modifications: Phosphorylation

Leads to Activation
[can change the net charge on the histone’s surface]

Histone tail modifications: Ubiquitination

Leads to Activation

What is the chromatin remodeling complex called?

SWI/SNF

p53’s effects and the consequences of its mutation

is a tumor suppressor → loss of function/mutation would potentially lead to uncontrolled tumor proliferation

p53 available(up) → p21 up → cdk2 down → RB up → E2F down → G1 does not go to S phase

(inverse is true for p53 mutation)

Different parts of RNA interference

RISC → RNA interference is done by this complex

Dicer → cleaves the DS RNA

Argonaute → cleaves between siRNA and mRNA

siRNA vs miRNA

siRNA is artificial

siRNA leads to translation inhibition and/or degradation

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miRNA is encoded by genome

miRNA leads to translation inhibition

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[Both are considered RNAi (RNA interference) and are involved in gene silencing]

[for a more practical example, RNAi is great for knock-out experiments in order to determine the functions of particular genes]

CRISPR-Cas9 3 steps

Acquisition - Cas9 locus → binds to virus DNA → GGG sequence → cuts 20 bases upstream

Expression - bait the DNA from the virus

Interference - Cas9 protein bind to CRISPR DNA

Requirements to accomplish CRISPR-Cas9

Required are Cas9(nuclease), Gene specific CRISPR RNA (crRNA), and tracrRNA → links crRNA to Cas9

What is the BEST strategy for genome editing among the 3 choices?

Zinc Finger / TALEN(helix-turn-helix) / CRISPR-Cas9

CRISPR-Cas9

Proteins Vs Nucleic Acids

20 amino acids vs 4 nucleic acids

Proteins have a large variety of functional groups

Proteins accelerate a multitude of chemical reactions

Proteins have well-defined tertiary structure (shapes)

why are there 64 combinations of nucleic acids

4 nucleic acids ^ 3 codon slots

highly degenerate code allows for the coding for every AA

genetic code modifications in the mitochondria

UGA = Trp, AUA = Met, AGA = Stop in mitochondria

tRNA cloverleaf secondary structure - all components

Amino-acid arm - conserved CCAOH → attaches to AA

Anticodon arm - read antiparallel to mRNA

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(of less importance below)

D-arm

Extra arm

TψC arm

what is “Wobble theory / third base wobble”

Adenosine at 5’ anticodon changes to I (Inosine) → can base pair with A, U, C

tRNA ala found to bind to GCA, GCC, and GCU → Inosine is the 3rd base

Inosine is highly ambiguous

Allows for more conservation of energy

Less stable interaction allows higher rate of protein synthesis

3D structure of tRNA

L shaped

Aminoacyl-tRNA synthetase does what?

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What does the process look like?

Matches tRNA w/ AA

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AA attacks ATP → becomes adenylated → Aminoacyl-AMP formed → 2’ OH of tRNA attacks C which releases AMP (which is a great leaving group)

aminoacyl-tRNA synthetase proofreading AA mechanism

AA too large → does not fit

AA too small → fits → proofreading site within enzyme → removed through hydrolysis

Ribosome History:

Who won the ribosome race?

_______________

Who first crystallized the ribosome?

_______________

Who won the ribosome race?

Tom Steitz

Who first crystallized the ribosome?

Ada Yonath

Ribosome binding sites and which subunit they are on

Large subunit

• E - Exit

• P - Peptide

• A - Amino

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Small subunit

• mRNA binding site

What are the rough percentages of RNA and protein in ribosomes?

What is the function of the protein in the ribosome?

2/3 (67%) of the ribosome is RNA and 1/3 (33%) is protein

Proteins are just there to stabilize the RNA

aa + ATP + tRNA → _______ + _______ ?

aa + ATP + tRNA → aa-tRNA + AMP (hydrolysis of ATP = loaded aminoacyl tRNA)

What modified AA is connected to initiator tRNA in bacteria?

Bacterial initiation utilizes fMet (formyl-Methionine) and initiator tRNA

fMet is loaded by 2 enzymes → Methionyl-tRNA synthetase & Methionyl-tRNA formyltransferase

Difference between fMet and Met tRNA is that there is a mismatch between AC which provides a kink !!!DIFFERENCE IS IMPORTANT!!!

fMet-tRNA to AUG (loads to P site) → regular methionine would load to A site