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Unit 6: Gene Expression and Regulation
Unit 6: Gene Expression and Regulation

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DNA

Deoxyribonucleic Acid (DNA):

A polymer made up of many monomers

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Nucleotide: (monomer)

Sugar (pentose, deoxyribose C5H10O5): 5 carbon sugar, in a pentagon shape

A ribose minus an oxygen

Phosphate Group (PO3)

Nitrogenous Base: what changes between nucleotides

Cing Tut == pyrimidines

Adenine (purine == double ring structure)

Thymine (pyrimidine == single ring structure)

Guanine (purine)

Cytosine (pyrimidine)

A always bonded to T and G always bonded C

DNA has two strands of nucleotides, and nitrogenous bases are held together by hydrogen bonds

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Antiparallel: DNA strands runs in opposite directions, one strand runs 5 to 3 and the other runs 3 to 5 (important for DNA replication)

5 Prime Side: there are five carbons (where the point of the pentose sugar points)

3 Prime Side: where three carbons at end of the pentos (fat side of the pentose points to the end)

DNA Backbone: sides of the nitrogenous base ladder rung structure, made up of sugar and phosphate, keep the DNA together

Double Helix: DNA twists around, made up of two strands of DNA

Proteins were considered genetic information for a while before discovering DNA, bec. There were 20 amino acids, which makes more sense for complex combinations than 4 bases

Frederick Griffith’s Experiment: mixed a rough strain of a disease (harmless) with a heat-killed smooth (virulent) strain and the mouse died

There was a transforming factor, from the heat-killed bacteria to the rough strain → DNA

Avery McCarty Macleod Experiments: wanted to discover Griffith’s transforming factor

Isolated the bacterial RNA, proteins, and DNA

Used enzymes that broke down each of those things until they could find out what caused the bacteria strain to transform

When they broke down DNA, they found that the bacteria could no longer transform → found life was based off DNA (not definitively)

Alfred Hershey and Martha Chase Blender Experiment: is it DNA or is it proteins that actually drive life (definitively)?

Dyed sulfur and phosphorus in a virus that invades a bacterial cell

Sulfur is found in proteins but not DNA

Phosphorus found in DNA and not proteins

Showed that the only one that was transforming the cell was the phosphorus → therefore DNA was hereditary material

Watson and Crick: credited for discovering DNA’s structure combining Chargaff’s ATGC levels and Wilkins and Franklin’s helical structure

Maurice Wilkins and Rosalind Franklin: discovered the crystallographic helical structure of DNA (basically had the data that Watson and Crick would use)

Erwin Chargaff: looked at the amounts of bases: ATGC

Found that A and T amounts correlated; G and C amounts correlated

Must be pairs: Chargaff’s Rule

DNA REPLICATION

during the S phase of interphase

Semiconservative Model (of replication): original/parental strands of DNA separate, and two new strands fill in with each parental strand. Each new copy of DNA is composed of one old parental strand and one newly made strand

DNA only dies if you don’t pass it down, “the immortal thread”

Every new DNA molecule is made up partly of old DNA

MESELSON AND STAHL Experiment: discovered semiconservative model

At the time, there were two other models of DNA that were proposed:

Dispersive Model: DNA was broken up, and each little DNA model was made up of old and new

Conservative Model: original parental molecule was conserved, and a two new strands were formed

Grew bacteria in heavy nitrogen, all DNA made in bacteria contained heavy nitrogen-15

Original bacteria has two strands of DNA both with nitrogen-15

Would have low band on centrifuge because of high density

Grew bacteria in nitrogen-14, all new DNA would be made using nitrogen-14 (lighter)

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Two new daughter strands made using nitrogen-14 but the old strands are all originally nitrogen-15

After first round of replication, each DNA strand was half 15 and half 14

Got a medium band on the centrifuge

Third Round of Replication

Centrifuge results:

Light Band: daughter strand of second round, paired with a new strand

Medium Band: old 15 band from original paired again with another new daughter strand

Never goes away because original nitrogen-15 DNA strand stays

Origin of Replication “bubbles”: different in prokaryotes vs eukaryotes

Where replication begins in each cell

Bacterial DNA is unicircular has only one origin of replication and extends outward in both directions around the circle until completely replicated

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Eukaryotic DNA: multiple origins of replication with linear strands of DNA

“Bubbles” at origins get bigger and bigger until they converge → multiple origins of replication make the process go faster and proves semiconservativeness

Contains two replication forks: extends outward at each fork

Creation of the Replication Bubble (made up of two replication forks)

DNA Helicase: responsible for opening DNA up and making the replication bubble → breaks hydrogen bonds of helix

Topoisomerase keeps theDNA unwound and stop hydrogen bonds from reconnecting “little clamps” DNA doesn’t want to stay apart

Single-strand Binding Proteins: keep the bubble open, and binds to the unpaired bases of DNA to stop them from rematching up

Making new DNA strands: (S phase of interphase)

Primase: not made up of DNA, made up of RNA, lays down a primerto begin DNA creation

5 to 3 == leading strand

Replication happens easily in the leading strand

3 to 5 == lagging strand

DNA Polymerase 3: the enzyme that actually makes the new DNA, can’t start from nothing, it can only extend preexisting nucleotides, needs to go off the primase’s primer

***can only operate in one direction ONLY operates to make 5 prime to 3 prime so it starts at the 3’ side of the leading strand to make a complementary 5’ to 3’, builds a complementary strand that’s opposite prime to opposite prime, by reading the template

Starts at the 3 prime side of the template original/parental strand to make DNA 5 prime to three prime

Then how do you make DNA for the other parental 5 to 3 strand but polymerase 3 can only go the opposite way

See DNA Replication Sheet on Drive

In the lagging strand, multiple not just one like leading strand, primers are going to be laid down, and polymerase 3 operates in between the primers

Okazaki Fragments: only found in the lagging strand, a segment where DNA is replicated in the opposite direction, but stops at each primer, and then moves to the left, then replicates to the right, and moves to the left…

P→ P→ P→

Overall direction: ←

All fragments are polymerased simultaneously, so it is quicker than just two primers on the end

Polymerase 1: removes the RNA primers and replaces with DNA

Ligase: connects fragments of DNA together, connects Okazaki Fragments to make a continuous strand of DNA

New daughter strands are IDENTICAL

If there is a mutation: Polymerase 3 screwed up

You get all of your materials from your food

Where do Polymerases get their nucleotides?

Nucleoside Triphosphate: an adenine nucleotide with two extra phosphates to which is ATP

Polymerase cuts off the phosphoruses and you get Adenine

Releases energy required to build DNA

There are also CTP, GTP, TTP, other energy sources that when phosphorylated become all of the other nucleotides in the body.

BUT, if at the very end of each DNA fragment, there’s nothing for Polymerase 1/3 to latch on to; end DNA is never replicated

With every single replication of DNA, your DNA gets shorter

Telomeres: contain non-coding DNA, long sequences of repetitive DNA at the end of each chromosome, it can be burned up through the process of DNA replication as to not affect your coding DNA

When telomeres run up, you die because you start eating into your genes when you replicate

Telomerase: enzyme found in germline cells (sperm/egg) that extend telomeres so you pass on full DNA

DNA REPAIR

If a mistake/mutation occurs, Polymerase 1 or 3 is at fault, by laying down the wrong nucleotides

We have enzymes that proofread our DNA and fix problems

“Mismatch Repair”/ Nucleotide Excision Repair: if nucleotides are mismatched, don’t line up properly, then a protein cuts out the bad nucleotides and fixes it

If this process were perfect there would never be any variation or mutations for evolution

Nuclease: DNA cutting enzymes, and it cuts out the mutation

Polymerase just reinserts the DNA

Ligase reconnects the DNA

***bacteria and viruses lack proofreading proteins, they want and need to mutate in order to evolve and create new strains

GENES AND PROTEINS

A gene is the instruction manual that creates a certain protein, each gene codes for 1 protein polypeptide

Beadle and Tatum Experiment:

Studied metabolic defects in a fungus

Mutated each specific gene that coded for a specific enzyme in a metabolic pathway and discovered that when a certain gene mutated a certain protein screwed up in the pathway causing a buildup of that product

Discovered the 1 gene 1 enzyme (now protein and now polypeptide) hypothesis

Every protein is different because it has a different sequence of amino acids

TRANSCRIPTION

: takes place inside the nucleus

DNA in chromosomes is too long and condensed, must use mRNA to leave through nuclear pores

Genes tell the ribosome what order to put amino acids in

One specific gene is read and used to make a sequential copy called mRNA (messenger RNA)

mRNA is a transcript/copy of a gene and goes to the ribosome

Gene DNA is read in triplets of bases called the reading frame TEMPLATE STRAND IS ALWAYS 3’ to 5’, the COPIED RNA CODING STRAND IS 5’ to 3’

RNA Polymerase 2: reads the template strand of DNA makes an RNA coding strand, which is complementarybut where there’s an A in the DNA, polymerase puts a U (uracil) in place of a T

The result is mRNA which in triplets has codons each codon corresponds to a specific amino acid

Determination of Structure/Function: ultimately the DNA determines protein structure

Sequence of DNA → mRNA (codons) → amino acids → protein structure → protein function

Proteins in different shapes perform different functions

AUG (start codon): codes for amino acid methionine, ALWAYS the first codon, and methionine is always the starting amino acid in every protein

Therefore, in DNA, the beginning of every gene is TAC

There are also stop codons, each gene always starts with a start codon and ends with a stop codon

Noncoding DNA: DNA that does not correspond to coding for a protein, it acts as a buffer, only 1% of DNA actually codes for proteins, the majority is noncoding

Most noncoding DNA is used to regulate our genes,

Promoter: region right before the gene of noncoding DNA where RNA polymerase binds to after transcription factor binds to TATA. Never transcribed as part of mRNA, only part of DNA. TURNING ON A GENE:

TATA Box: first four nucleotides of promoter (TATA)

When a transcription factor binds to the TATA Box, begins the process of transcription.The transcription factors (steroid hormones) are what turn genes on. Steroid hormones control the process of protein synthesis

Terminator Sequence (AUAAA): a sequence that causes the RNA polymerase to stop transcription and send off the mRNA

mRNA transcription starts at the promoter and ends at the terminator sequence

Before mRNA can leave the nucleus, the Pre-mRNA must go under RNA Processing: stuff added to the Pre-mRNA before leaving the nucleus

5 Prime Cap: made out of GTP, how the ribosome knows what part of the mRNA is the front

Poly A tail: long string of adenine, that protects against degradation (back of mRNA)

RNA Splicing:

Exon: parts of the RNA are coding, each exon corresponds to a location in a polypeptide called a domain

Intron: part of RNA, that is noncoding

Alternative Splicing: why have introns when you could just have exons?

23,000 genes can actually code for more than 23,000 proteins

One piece of mRNA can code for multiple proteins in the body by switching the exon and intron parts

Spliceosome: cuts out the introns and splices together the exons so only coding mRNA is left

TRANSLATION

: comes after transcription in ribosome

Ribosome receives and reads the mRNA and reads it, puts together a sequence of amino acids

Redundancy of codons: if a mutation takes place you may not put down the wrong amino acid, protection against mutations

Multiple codons can code for the same amino acid

Silent Mutation: mutation that doesn’t do anything, doesn’t change the amino acid it codes for, because of redundancy

tRNA: (transfer RNA) bottom part of the tRNA is a complementary, anticodon that is complementary to the codon (STILL USES URACIL) and carries an amino acid on the top. tRNAs can be recycled, just attach new amino acids to build a protein

Each has a specific anticodon and amino acid

Aminoacyl-tRNA-synthetase: enzyme that attaches tRNA to new amino acids, using ATP (get amino acids from food)

Ribosome Structure: (GTP powers everything) CONVEYOR BELT

Binding sites:

Small Subunit: scans mRNA for the start codon, the mRNA lays on top of the subunit and tRNA attaches

Large Subunit: (Left to Right: Environmental Protection Agency) attaches on top of small subunit

E Site: exit site, where the used tRNA leaves

P site: first tRNA enters

A Site: next tRNA enters

Works like a conveyor belt, tRNA attaches to the codon

SEE THE ANIMATION ON GOOGLE CLASSROOM

Release Factor: carries water and performs hydrolysis, enters the A site instead of another tRNA and stops the building of a protein

GTP used to dehydration synthesize the amino acids, creating peptide bonds and GDP

Polyribosomes: Since it’s a conveyor belt, you have multiple ribosomes working on the same strand of mRNA, speeds up the rate of protein production

Bound Ribosomes:

Protein Structure:

Primary Structure: linear sequence of amino acids peptide bonded together

Protein folding takes place in the endoplasmic reticulum

Secondary Protein Structure: hydrogen bonding creates beta pleated sheets and alpha helices

Tertiary Protein Structure: Hydrophobic bonds, ionic bonding, disulfide bridges, etc. creates one polypeptide subunit

Quaternary Protein Structure: multiple polypeptides come together to form a complex protein

Wobble: for each amino acid, there is only ONE tRNA that carries it, even though there are multiple codons that code for a single amino acid (redundancy), only the first two nucleotides of codon and anticodon fit together, the last one doesn’t usually matter, so the tRNA and mRNA fit wobbles, not perfectly together to save energy

Relaxation in the base pairing rules for the third nucleotide

Eukaryotes vs. Prokaryotes:

RNA processing only takes place in Eukaryotic cells (prokaryotes don’t have spliceosomes)

Prokaryotes don’t have a nucleus

MUTATIONS

Mutations take place during the process of DNA replication (polymerases screw up)

Point Mutation: single base modification causes a nucleotide base change, insertion, deletions, substitutions, etc.

Substitution: one nucleotide is substituted for another (Sickle Cell Anemia)

Changes only one amino acid in a protein, can radically change the shape of a protein (change takes place in the primary structure, and all other structures, etc.)

Tay Sachs: lipase in the brain (breaks down lipids) mutates so lipids in the brain build up and kill the baby

Missense: when you have the wrong nucleotide and it changes the amino acid

Nonsense: when the mutation creates a premature stop codon (bigger effect on the protein than missense)

Silent Mutation: change at a point that doesn’t change the amino acid because of redundancy (third nucleotide usually doesn’t matter)

Insertion & Deletion:

If a nucleotide is put in where it shouldn’t or if a nucleotide is removed.

Causes a shift in the reading frame, causes all triplets to be screwed up

Frameshift: all reading frames moves

Affect every amino acid after that mutation

CONTROL OF GENE EXPRESSION

Not all genes are turned on all of the time, else you would be making all of your proteins constantly which would waste resources and energy.

Cells regulate genes to control the production of proteins in the body using hormones (mainly steroids)

Turning on genes causes:

Create different proteins

Create different cells

Development (changes that take place during your lifetime, puberty especially)

If a gene is being expressed it means its being transcribed or turned on

Cell Differentiation: Different types of cells (heart cells, skin cells, etc.) are created by turning on and off certain genes even tho every cell has the same code

Happens in the womb

Stem Cell: a cell that has not yet differentiated

Differential Gene Expression: having certain genes on or off

Internal and External Factors can affect gene expression

Operons and other structures herein are only found in prokaryotes, not eukaryotes, a simplified model

Operon: a region consisting of the operator, genes, and promoter

Operator: only in prokaryotes, sits in front of RNA polymerase before gene and after promoter, controls whether or not genes are turned on or off

Regulatory Gene (way before operon): codes for the repressor protein which can bind to the operator, if the repressor binds to the operator, RNA polymerase cannot get to the genes → turns off the genes/inhibits gene expression or transcription

Repressible Operon: normally turned on but can be turned off

Trp (tryptophan) Operon

EXAMPLE: Leads to the production of tryptophan proteins

The bacterium will turn off the tryptophan if it consumes enough of it

The repressor is normally the wrong shape so it cannot bind to the operator to shut off the gene, so tryptophan acts as a corepressor, binds to the repressor changing its shape. The new structure can now bind to the operator and shut off production of that gene → don’t waste energy making more of the protein when you have enough

Turned back on when you run out of tryptophan because the repressor turns back into its inactive shape

Inducible Operon: normally turned off but can be turned on

The lac operon (stands for lactose sugar found in milk)

Produces lactase enzymes needed to break down lactose

Only want to produce these enzymes when you ingest lots of lactose/milk

The repressor is normally bound to the operator, RNA polymerase normally does not have access to the genes → turned off most of the time

Lactose is an inducer changes the shape of the repressor to release it from the operator, and makes it inactive

If there’s no more lactose it would shut off the gene

EUKARYOTIC GENE CONTROL

Eukaryotic Genome:

70% of the genome occurs only once 1% is actually coding the other 69% is used to regulate your genes

30% is repetitive

Control Elements: regions where transcription factors (steroids or growth hormones NOT just roids, etc.) bind, noncoding regions

Promoter

Proximal Control Elements: close to promoter

Distal Control Elements (enhancers): far from promoter

Transcription Factors:

Activators: turn genes on

Usually bind to the enhancer/distal region

Repressors: turn genes off

*Can delay/stop the process of protein production is to not process the Pre-mRNA

Noncoding RNAs: used to slow or stop the production of proteins by blocking or destroying mRNA before they get to the ribosome, instead of turning off the gene → regulate protein synthesis

Coding RNAs are rRNA tRNA and mRNA used to make proteins

MicroRNA (miRNAs)

Small interfering RNAs (siRNAs)

Interference: prevents mRNA from hitting the ribosome put in a holding pattern

Degradation: destroy mRNA

Proteases break down extra proteins in a proteasome to purely destroy unneeded proteins

Modifications to Chromatin Structure

Control whether genes are on or off by changing chromatin structure

Either loosen or contract DNA to expose or hide genes for replication, if hidden, polymerase cannot get to the DNA

Histone modifications

Acetylate histones: expands chromatin by attaching acetyl groups to the histones

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Unit 6: Gene Expression and RegulationUnit 6: Gene Expression and Regulation

DNA