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Katie Nash Bio Exam 3, Review Guide

Chapter 11

CLASSIC EXPERIMENTS IN BIOLOGY

Classic Experiment #1: Griffith transforms bacteria (1928)

The central Dogma is not yet known

Griffith looks at two strains ofstreptococcus pneumoniaeo R strain: harmlesso S strain: pathogenic has a polysachharide coating that protects the bacteria from

the mouses immune system, will kill the mouse

Heated the S strain and it is no longer pathogenic in the mouse, denatured the DNA thatcodes for the protein to grow the protective coating But when the heat killed S strain mixes with the live R strain the mixture become

pathogenic the R strain was somehow transformed by heat killed S strain; for R strains

with the protective coating, the R strain had taken up a piece of the S strains DNA

Bacteria can only reproduce asexually; transformation is one way for them to exchangegenetic material

Refinement of Experiment #1 Avery, McCarty and MacLeod(1944)

Asked what macromolecule was the genetic material or the material that transformed themouse

Separate the heat killed S strain into proteins, lipids, and nucleic acid components Demonstrated that mixing just the nucleic acid component with the live R cells

transformed them but nucleic acid component still had some contamination- not allscientists were convinced

***not sure which experiment is right

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Put S strain bacteria in three test tubes one with RNase, one with Protease and the other

with DNase then mixed it with the R strain

The tube with DNase was the only one that was no longer infectious; therefore DNA is thegenetic material

Classic Experiment #2: Hershey Chase, 1952 Bacteriophage: viruses that infect bacteria and only bacteria, do no infect human cells

o Lunar landing modules; protein coding, interior has DNAo Sits on the surface of the cell and injects bacteria with its DNA, DNA codes for more

phage, and makes the cell into a virus producing factory

They knew about the chemical composition of protein and nucleic acids and knew thatSurfur is found in some proteins but not in nucleic acids and the phosphorous is found in

nucleic acids but not in proteins

Attached radioactive sulfur and phosphorous labels to virus and put them in a beaker withbacteria

Centrifuge so that the less dense material is the bacteria without the virus and the denser isthe bacteria with virus material the pellet had radioactive phosphorous and very little

sulfur which suggested that DNA is the genetic material that viruses transfer

Hersey wins the nobel prize for this experiment in 1969

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Eukaryotic cells can also be transformed with DNAo Can be done with plasmids, small linear molecules of DNAo In nature bacteria are always exchanging DNA

Classic Experiment #3: Watson and Crick, 1953

Watsons idea- think that DNA is the holy grail; not everyone convinced yet Did not know the structure or connectivity of DNA Watson and Crick combine many other peoples work to put together a more complete

picture of DNAs structure

The Key data for Watson and Cricko 1.Chargraffs rules: the # of G= # of C and # of A=# of T; knew that they bonded

together- base pairing

oo 2.Franklins X-ray crystallography: images show a helical repeato 3.Phosphate sugar backbone on the exterior had built a model with phosphate on

the interior but talks to Franklin and realize to put charged phosphate on the

aqueous side

o 4.Positive hydrogen bonding between bases talk to chemists about how bases willinteract; were originally looking at the wrong representation of guanine

o three bonds between G and C; two between A and T suggested that it would be easier to undo bonds between A and T

Co-publish with Rosalin Franklin Suggest in their paper how replication might take place

Classic Experiment #4: Meselson and Stahl, 1958

Asked how does the cell make an exact copy of its DNA? Are the original strands used astemplates?

Three possibilities for how replication occurs:

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o Semiconservative: one original strand is the template for one new strando Conservative: complete replicationo Dispersive: part of new and old mixed in together

Tag light and heavy DNA; originally only heavy and then put light nucleotides in themixture and allow the DNA to replicate the density of the substances tells how it had

replicated

Parent generation: only heavy First Generation: intermediate one heavy and one light strand; heavy disappeared-

against conservative

Second generation: partially intermediate, partially lighteach of the strands from the firstgeneration had served as a template; against dispersive- not all intermediate

1956 Arthur Kornberg isolates the enzyme capable of polymerizing DNA in vitro

DNA polymerase What you need to make DNA

o All four dNTPs (nucleotides): aNTP, tNTP, cNTP, gNTPo DNA fragment (template and primer)o DNA polymeraseo Mg2+ -- masks the negative charge on the phosphate groups

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Through a messy preparation discovered thatprimer was necessary for DNA polymeraseo RNA polymerases do not require a primero Primase: a specific RNA polymerase that makes the RNA primer for DNA replication

REPLICATION

Directionality of Replication:

o Nucleotides can only be added to the 3 end of the DNA strando Phosphate group is attached to the 5 carbon and OH group is attached to the 3

carbon

o ATP, GTP,TTP come in to form the phosphodiester bond with the next nucleotidein the chain. The energy for the formation of this bond comes from the breaking off

of two of the three phosphate. where we get the energy for this otherwise

entropically disfavored anabolic processo If the dNTP came in on the other side there would be no phosphate bond to break

and no energy to supply the bond formation

o This also explains why we need a primer for DNA polymerase cannot hydrolyzethe phosphate bond and obtain energy needed to bond nucleotides RNA

polymerase has a way to do this

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The replication fork: chromosomes are long and we cannot unzip the entire double helixall at once, takes too much energywant to reassociate; so we unzip part of the helix at a

time forming a replication fork

o Read nucleotides on the template from 3 to 5o Build nucleotides on complement from 5 to 3o the lagging strand: DNA replication is discontinuous: need to build 5 to 3 but

cannot on one side of the replication fork; create many different primers to build 5

to 3; when DNA polymerase reaches the primer from before, falls off and startsagain; as we unzip more we add more primer

o DNA ligase: joins together the Okazaki fragments; while DNA polymerase Ireplaces the primers with DNA

Summary of Events: Prokaryotic Replicationo 1. Helicase unwinds the double helix using energy from ATPo 2. Single Stranded binding proteins stabilize the unwound helixo 3. Primase: makes RNA primers on both the leading and laggings strandso 4. On the leading strandDNA is synthesized continuously 5 to 3 towards the

replication fork by DNA polymerase III

o 5. On the lagging strandDNA polymerase III elongates Okazaki fragments from theRNA primers; elongation ends when pol III hits the previous primer

o 6. DNA polymerase I removes the RNA primer and replaces it with DNA; it is aexonuclease: can remove nucleotides

o 7. DNA ligase: bonds the space left between what used to be an RNA primer and theend of the adjacent strand; requires ATP

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The replication fork: chromosomes are long and we cannot unzip the entire double helixall at once, takes too much energywant to reassociate; so we unzip part of the helix at atime forming a replication fork

o Read nucleotides on the template from 3 to 5o Build nucleotides on complement from 5 to 3o the lagging strand: DNA replication is discontinurous: need to build 5 to 3 but

cannot on one side of the replication fork; create many different primers to build 5

to 3; when DNA polymerase reaches the primer from before, falls off and startsagain; as we unzip more we add more primer

o DNA ligase: joins together the Okazaki fragments; while DNA polymerase Ireplaces the primers with DNA

In Eukaryotic Replication, Multiple Replication forks speed up the process: ligaseconnects the pieces

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Eukaryotes have proof reading mechanisms that prokaryotes dont haveprokaryotesdont really care because they replicate so fast and so much

Ave. Length: length of replication bubble (kb- kilabases) Proof reading mechanisms

o If bases stick correctly, they will stay, if not they usually fall offo DNA polymerase III has proofreading mechanism recognizes changes in width-

should be uniform

DNA proofreading: takes place during replication Mismatch repair mechanism scanned DNA immediately after it ahs been replicated and

corrects any base=pairing mismatches

o After DNA has been replicated a second set of proteins look for mismatched pairs these proteins excise the mismatch base and adjacent bases

o Knows the mismatch by the size in the DNAo DNA polymerase I adds the correct bases back in

repair mechanism removes abnormal bases that have formed because of chemical damageand replaces them with functional bases

o Deal with damage from chemical reactions that can damage DNAo Excision repair proteins: excise the damaged base and some adjacent baseso DNA polymerase Iadds the correct bases by 5-3 replication of the short strand

TELOMERES

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Telomeres: the ends of chromosomes (telos in greek- end; meros- part) the ends cannot be left rawbecause they would then be recognized by repair mechanisms

and fused with other sequences; would also be subject to enzymes that protect against

foreign DNADNAases, RNAses- job is to break down nucleic acids

Instead there is a long repeating sequence that folds over onto itself rich in guaninescanmake H bonds with other guanines

Experiment shows function of telomeres:o some artificial chromosomes with and without ends are added to yeast cells;

without the ends they start to get chewed up by enzymes

Telomeres and DNA replicationo When RNA primer closest to the linear end is removed DNA polymerase I cannot fill

the gap because it had no primer to work with

o There will be a part at the end that is single strandedo leads to Telomere attrition: shortening of telomeres after series of replications-

after a while the cell di

telomerase: enzyme that replicates telomeres or chromosome ends contains a piece ofRNA that acts as a primer

o most cell types do not express telomerase the symptoms of aging have beenconnected with the shortening of the chromosomes

o but turning on telomerase in the wrong cells makes rapidly dividing cells immuneto the signals that recognize rapid division as bad- the signal is shortened

telomeres- no more apoptosis in this way

o cancer cells express telomerase as one way to confer immortalityo telomerase is an interesting target for cancer drugs

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Chapter 12

DISCOVERING THE CENTRAL DOGMA

Early on thought that one gene one enzyme, but not all proteins are enzyme Then one gene one protein?, there are quaternary subunits so not exactly Now we think **ONE GENE ONE POLYPEPTIDE; however this too is at fault because some

genes code for RNAs and then never become a protein they stop in the middle of thecentral dogma

Archibold Garroda physician in London who had several patients who suffered fromblack urine disease; the disease appeared to be inherited through families called itin-

born errors of metabolism

o 1909 he proposes that the disease resulted from the inability to make a certainenzyme something should be metabolized before it gets to the urine, but it is not

in this case

o accumulation is problematicClassic Experiment #5: Beatle and Tatum

wild type Neurospora (bread mold haploid life cycle) can survive on a minimal medium(contains inorganic salts, glucose, and biotin)

treated neurospora with light x-rays so that they can no longer grow on minimal mediado this many times to produce varying mutations

identified mutants that can survive on a complete media (media supplemented with all 20amino acids and other nutrients) but not on a minimal media the mutants had lost the

ability to synthesize a single amino acid

gave the neurospora a complete media without the amino acids and then a complete mediawithout the vitamins the mutant does not need the vitamins but needs the amino acids

now want to know which amino acid is the problem? Figure out that they only need to addargenine (grew a sample on minimal medias each with a different amino acids)

found three different genes mutations that required the addition of exogenous argenine

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discover that enzymes work in pathways mutations affect different steps in the pathway one gene to one polypeptide

the prokaryotic central dogmao no nucleus in contrast to eukaryotes where transcription occurs in the nucleus and

translation occurs in the cytoplasm or on the RER;

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o viruses are an exception to the central dogma some viruses have DNA and some have RNA the advantage of RNA is that they can start translation the moment they

enter a host cell

retroviruses make DNA from RNA insert it into the nuclear DNA; we donot have the mechanisms to undo this

TRANSCRIPTION

Initiation: 1. RNA polymerase binds to a promoter;

o Promoter: specific sequence before protein coding that signals the transcriptionmachinery

there are fewer promoter sequences than genes a series of genes might have the same promoter sequences and then all

genes in that pathway might be transcribed simultaneously

nucleotides upstream of the initiation site help RNA polymerase bind recognition sequence: sequence recognized by RNA polymerase TATA box closer to the initiation site: rich in AT parirs- point at which the

DNA begins to denature so that the template strand is exposed

o in replication the initiation site is the orino termination site; 2.transcription begins at the initiation site- a part of the promoter where transcription

begins

o in transcription we do not make proteins for the entire genome unlike replication we replicate the entire genome at once

3. Conformational change in the RNA polymerase denature a short segment of DNA creatingan open complex ready for base pairing

Elongation: 1. RNA polymerase unwinds the double helix 10-20 bp at a time reading the template from

the 3 to 5 end

o RNA polymerase does not require a primer 2. Adds ribonucleotides to the 3 end of the growing strand

o does not proofread or correct work (unlike in replication 3. DNA double helix rewinds as RNA polymerase moves through

o this rewinding requires energy supplied by RNA polymerase and the break down ofpyrophosphate molecules

Termination: 1. a particular base sequence signals to terminate transcription 2. DNA polymerase separates from the templateComparing Transcription and Replication

Replication transcription

How nucleotides are added To the 3 end using energy

from the phosphate bond to

create the phosphodiester

bond

same

How the template is read From 3 to 5 same

Enzymes involved DNA polymerase III, DNA poly

I, II, ligase, helicase, primase

RNA polymerase

How much template is used Entire genome Small portion of the genome

Which nucleic acid is made DNA RNA

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Is a primer required Yes No

How do we know where to

start?

Origin Promoter

How do w know when we

reach the end of the process

End of the genome ter Termination site

TRANSLATION

the genetic code: with 3 letter words there are 64 possible codonso tripletso this means that the code is redundantbut not ambiguous

third base in a codon is often not specificwobble- not picky in the thirdposition; if we are picky it is between purines and pyrimidines

silentmutations are often at the 3rd nucleotide- wont make a difference intranslation

o contains stop and startcodonso nearly universal across specieso explains how a single nucleotide change can result in the single amino acid

substitution

oo put all one nucleotide in the beaker and requirements for translation

tRNA: the adapter molecule- bridge between mRNA and protein

in the absence of biological evidence Crick postulated than an adaptor molecule must be

involved in the translation of nucleic acids into proteins

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the stem like 3D structure of the tRNA molecule allows it to bond to both an amino acid andthe mRMA transcript as well as have a unique structure

o this structure is formed by hydrogen bonding between tRNA base pairso also hydrogen bonds with mRNAo has an anticodon that corresponds to a particular codon on the mRNAo the other end is the amino acid attachment site the amino acid binds to the

hydroxyl group on the end

Charging tRNAs through the Amino Acyl tRNA synthaseo There are specific enzymes for specific tRNAs and specific amino acidseverything

is lock and key fit this ensures that the right amino acid is attached to the right

tRNA; then the codon/anticodon makes sure that amino acids are added in the right

order

oo the synthase reads the anticodon and adds the right amino acid

The ribosome: made up of RNA and proteinrRNA (there is also mRNA, tRNA, mtRNA)o Large and small subunitso 3 binding sites or pockets in the large subunit in which the tRNA will bondo when not involved in translation the two sub units exist separately from each other

Initiation of Translation:o 1. Small ribosomal subunit binds to the recognition sequence on the mRNA

in eukaryotes there may be a sequence before the AUG start codon, but notin prokaryotes

o 2. Metionine charge tRNA binds to the AUG start codon to complete the initiationcomplex (tRNA + small sub unit + mRNA)

o 3. Large ribosomal sub unit joins the initiation complex with methionine chargedtRNA occupying the P site

o initiation factors: proteins that put mRNA, ribosomal subunits, and metioninecharged tRNA together

o there will be an overhang of info before the start codon that does not get translated

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Elongation

o 1. Charged tRNA whos anticodon is complementary to the 2nd codon binds to thiscodon at the A site

o 2. The large sub unit catalyzes two reactions demonstrating peptidyl transferaseactivity

a. breaks bond between the tRNA in the P site and the amino acid on thattRNA

b. forms a peptide bond between the two amino acidschain grows on thetRNA in the A site **the carboxyl of methionine joins the amino groups of

the next amino acid

o elongation factors: proteins that assist in elongationo 3. Translocation of the ribosome so that a tRNA enters the E or ejection site and

others shift overejected tRNA goes back into the soup to find another amino acid

and synthetases

o 4. Proceeds until it reaches the stop codono ** elongation uses GTP instead of ATP not really important whyo

protein folding commences right away Termination:

o 1. Release factor binds to the A site when the stop codon comes upo 2. The release factor disconnects the polypoptide from the tRNA at the Pside via a

hydrolysis reaction

o 3. Remaining components of the complex separate polysome: more than one ribosome on the same mRNA; ribosomes are not stable, they

keep moving; as soon as there is space for a new ribosome to show up it will

**in prokaryotes translation can begin as soon as the 5 end of the mRNA is available- somultiple ribosomes can be translated at a time while the mRNA is still be transcribed one

mRNA can make many proteins not true in eukaryotes

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Transcription Translation

Initiation signaled by: Promoter sequence in the

DNA

Start codon in mRNA

Termination signaled by: Terminator sequence in DNA Stop codon in mRNA

WHAT HAPPENS TO PROTEINS AFER THEY ARE SYNTHESIZED ON THE RIBOSOME

Signal sequence in a protein (not mRNA)o 1. Says to continue translation on the free ribosomeo 2. Go to the RER im for export or a membrane proteino factors in the cytosol recognize these signals on the amino acids

the SRP: signal recognition particle binds to the signal sequence and the ribosomeslowing down translation

o if the signal says to go to the ER the SRP takes the ribosome to ER by binding to asignal receptor near the translocation complex on the membrane of the RER

o the signal sequence is handed over to the channel and the binding of the signalsequence to the channel opens the channel

o the SRP then leaves the ribosome and the translation process resumes through thechannel

o protein is threaded through the lumen of the ER or is wrapped around themembrane if it is a membrane protein hydrophobic sequences get retained in

the membrane and threaded so that hydrophilic sequences are on the outside

o ** otherwise the signal sequence sends the protein to an organelleo SPC signal peptidase complex removes the signal sequences from the peptide

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POST-TRANSLATIONAL MODIFICATIONS

Proteolysis Glycoylation PhosphorylationMUTATIONS

Mistakes can be made during transcription but they are less serious because the mistake isonly in one mRNA transcript; still, we use one template many times- cause many faulty

proteins

Silent mutation: point mutation that does not make a difference in translation Missense mutation: change the identity of a triplet that results in the wrong amino acid

being madestill may not be of any real consequence if its not the active site or an

important part of the proteinbut also could be

Nonsense mutation: a premature stop codon and shortened protein results from thismutation; odds of having functionality is unlikely

Frameshift mutation: messes up the reading framemay lead to a missense or nonsenselike mutation or both; only the position of the start codon tells the ribosome how to read,

from there its on its own; multiples of three wont be so bad

Induced mutations: mutations from mutagens changes the chemical nature of the baseso that it will bond incorrectly

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Chapter 13

VIRUSES

What is a virus?o A virus is a delivery system for the viral genomeo Viruses can only replicate after they successfully infect a host cello The outer part of the virus is packaging for the viral genomeo An infected cell because a virus factory which in turn leave the cell and move on to

infect another

o A virus is NOT a cell Appearance of viruses varybacteriophage; herpes, etc.

tobacco mosaic virus: infect plants large helical genome in the center; protein coating is

shed

adenovirus: very common, has unique glycoproteins and a protein capsid influenza virus: also has glycoproteins that determine its identity (H1N1 for example);

enveloped virus

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bacteriophage: head; tail sheath and tail fiver injects DNA Genome: made up of either DNA or RNA; each can be double or single stranded Infection: have sabotage mechanisms to shut down the host cell depends on the virus Inside the capsid: proteins and nucleic acids Use host cells machinery- RNA polymerases, etc to reproduce themselveshave high

affinity promoters

The Viral Life Cycleo Obligate intracellular parasites: which means they only reproduce inside a hosto Host range: each virus has a limited number of cell types that it can infect; ex. HIV

infects T cells, flu infects respiratory cells

o Viruses use enzyme, ribosomes and other cellular material to synthesize progenyviruses

Animal Viruses can be DNA, RNA, single stranded, double stranded; may or may not havea lipid layer

Enveloped viruses: derive lipid layer form the plasma membrane;o note that underneath the envelop is the normal capsid layerproteino the nucleic acid has to get out of two layerso must be strong enough to protect the virus but also destructable enough to break it

down and release the nucleic acids

o this breaking down usually occurs in the lysosome Life Cycle of an Enveloped Virus

1. Enveloped virus (with its own lipid layer) attaches to the cell through its glycoproteins 2. Fuses with plasma membrane and enters the cell not like phage, no injection 3. Makes copies of its RNA in the form of mRNA skips the step of DNA RNA

o in this case we have a RNA dependent RNA polymerasethis comes from thevirus because we dont have that (only DNA dependent)

4. mRNA transcripts are translated into viral proteins capid proteisn, glycoproteins formembrane, etc.

o some viruses have early genes and late genes: early genes are transcribed torpoduce essential proteins for the remainder of transcription and translation

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5. New virion is constructed and it fuses with plasma membrane of host to be release fromthe cell

Alternative Life Cycle of an Enveloped Virus 1. receptors on the cell recognize the glycoproteins on the outside of the virus; the virus

attaches to the cell and the cell engulfs it via endocytosis

2. Now inside the cell in a double membrane. Viral and cell membrane fuse to releasecapside and viral genome

3. Transcription and translation of viral proteins and construction of new virus to bereleased by cell- membrane of the virus comes from plasma membrane of the cell

exocytosis

viruses have not evolved to check for errors- dont need identical replication this is a

chance for adaptationwhy we need a cocktail of drugs for HIVit has adapted antiviral drugs

o have tried to block virus from entering the cellcould not do ito can inhibit RNA dependent RNA polymerases- needed by the virus but not by the

cell used in HIV drugs

o also targetreverse transcriptaseHIVo but it is hard to do because all the enzymes are so similar- you dont want to target

human enzymes ex. AZT a nucleotide analog doesnt always interact withreverse transcriptase; cancer cells originally tried to target enzymes in cells that are

rapidly dividing

Life Cycle of a Retrovirus Ex. HIV: enveloped virus with viral RNA; undergoes reverse transcription; HIV has a copy of

reverse transcriptase

1. Glycoproteins recognize CD4 protein on the host cell 2. Fuses with membrane of the host cell to enter the host cell 3. Reverse transcriptase makes a cDNA strand the complementary DNA strand to the

viral RNA; then RNA is broken down

4. cDNA acts as a template for a second complementary DNA stranddouble stranded 5. Viral DNA must enter nucleus of cell- tough job

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6.Viral DNA is integrated into the nuclear DNA, known as a provirus (what is integratedinto host DNA) our genome has many different retroviral sequences but they have

mutated over time so that possibly promoter sequences are no longer recognized- now

considered junk DNA

7. Hosts RNA polymerase transcribes proviral DNA into RNA molecules 8. Translation of mRNA transcripts makes viral protein

o viral protease: when viruses make proteins they make them in one fell swoop asopposed to in pieces

polyprotein is made and in order to be active needs to be cleaved by viralprotease

used as a target for drugs- because isnt used by the host cell

BACTERIAL GENETICS

Replicate via mitosis; random mutations introduce changes, but also can exchange geneticmaterial between each other

Conjugation: bacteria sexo Observation: when two nutrient requiring strains are grown together a few bacteria

that are no longer nutrient requiring can be isolated

o Conclusion: bacteria can exchange genetic materialo F+ can form sex pili and F- cannoto Transfer genetic material through the conjugation tube: only attached for a certain

amount of time- only a short segment of DNA was move through- can send part of

the genome or a plasmid

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Transformation:

o Observation: Griffiths experiment with the mouseo Conclusion: bacteria can take up bits of genetic material from their environmento Transformed DNA can remain as a plasmid or can be incorporated into the other

genome

o Transduction: bacterieophage accidently packages DNA from one strain and delivers it to

another

o when the capsid forms in the infected cell it does not differentiate between viral andhost DNAsometimes host DNA gets inside

o when that virion infects a new cell it will have old bacterias DNAo this process is important to bacterial resistance to antibioticstransform the

resistance factor

plasmids: small circular chromosomes that are present in addition to the mainchromosome

o convenient way to deliver genes to bacteria for research

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o bacteria can exchange plasmids during replication, some plasmids contain antibioticresistant genes

GENE REGULATION IN PROKARYOTES

affinity of the promoter sequence: In prokaryotes the affinity of the promoter for the RNApolymerase determines how frequently this gene will be expressed- or how frequently RNA

polymerase will bind to it

o In eukaryotes the promoter also is essential for gene regulation and expression butthe polymerase enzyme in eukaryotes cannot bind to the promoter on its own. It

requires transcription factors to help it bind to the initiation complex

o Other regulatory proteins also bind to DNA sequences close to and far from thepromoter that also stimulate or repress transcription

o The presence and availability of these proteins is what regulates transcription ineukaryotes

An inducible system: requires an external signal to induce the gene expression; not alwayson

o Ex. Lactose breakdowno When lactose is absenta repressor regulatory molecule is bound to the DNA

blocking RNA polymerase

o In the absence of lactose, the repressor is boundthis is its default stateo When lactose is present: repressor is released from the DNA lactose acts as an

inducer that binds to the repressor so that it becomes inactive

o When lactose is present- the genes will be transcribed and the transcript will betranslated

A repressible systemo ex. Trp Operon gene for the production of TRPo at certain concentrations Tryptophan binds to the inactive repressor which makes it

able to bind to the operator

o so when there is no Trprepressor is inactive and when there is Trp repressor isactiveno transcriptiongenes for trp biosynthesis are turned off

inducible system: default is off; presence of inducer turns genes on repressible system: default is on, presence of repressor turns genes off How Inducible and Represssible systems are affected by mutation

o DNA mutation at the binding site of a repressorgenes are always expressed

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o Mutation in a specific gene- mutant enzyme is madeChapter 14

COMPARING PROKARYOTIC AND EUKARYOTIC GENES AND GENOMES

**Eukaryotes have lots of DNA that does not code for protein; ex introns, operators, etc.

in Eukaryotes transcription and translation are separated- transcription occurs in thenucleus

Eukaryotes have additional control sequences: enhancers, silencers In Eukaryotes the mRNA exists for longer in the cell due to capping and tailing which

stabilizes mRNA

comparison of a simple prokaryote vs. a simple eukaryote

Metabolic pathway genes are the same

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What makes the difference in the length is..o The number of genes needed for DNA replication and repair- due to things

like proofreading that eukaryotes do but prokaryotes do not

o More genes for regulating transcription and translationo Also needs more proteins for structure- compartmentalization

Multicellular eukaryotes

Genes for transcription control tells each cell how to differentiate- turns genes onand off

RNA processingcreating tissue specific mRNA The tissue formation and cell-cell singaling is not relevant in unicellular

organisms

CENTRAL DOGMA FOR EUKARYOTES

Eukaryotic transcriptiono Both eukaryotic and prokaryotic genes include promoterso The terminator sequence signals the end of transcription *** it is not

transcribed

Introns and exons: eukaryotic phenomenon-o the pre-mRNA is the unprocessed mRNA which is not yet ready to be translated

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nucleic acid hybridization: used to find intronso target DNA is denatured to break the hydrogen bonds between base pairs and

separate the two strands

o put in a single stranded probe if the probe had a base sequence complementary tothe target DNA, a probe-target double helix forms by hydrogen bdongin between

basses- but if they do not match up loops will formstretches of DNA that does not

have complementary bases on the mature mRNA

o studies of pre-mRNA later- showed that introns were part of the pre-mRNAtranscript

REGULATION OF EUKARYOTIC GENES

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REGULATION OF TRANSCRIPTION IN EUKARYOTES initiation: presence or absence of certain transcription factors

o **the operon is specific to bacteria and prokaryotes, not eukaryoteso in prokaryotes- promoter consists ofrecognition sequence and TATA boxo in Eukaryotes: RNA polymerase II cannot bind o the promoter without

transcription factors (TF)requires many before it can bind and then morebefore it begins to transcribe

o TFIID: first transcription factor

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enhancers: bind activator proteins and this binding strongly stimulates the transcription

complex

silencer sequences: like activator but repress transcription regulatory sequences: upstream of the promoter- regulatory proteins can bind to it

looping = seen in electron micrographs ***only unzips after everything has bound proteins that bind to the DNA can bind in 4 differentmotifs

o the protein side-chains all interact witht eh chemistry of bases and DNA- bind tothe major grooves

o helix-turn-helix developmento helix-loop-helix immune systemo zinc finger steroid hormone receptorso leucine zipper cell division

chromatin remodeling: nuelceosomes block both initiation and elongation steps oftranscription; too free the DNA a remodeling proteins binds and disaggregates the

nucleosome so that the transcription complex can bind and RNA polymerase can begintranscription; a second remodeling protein binds once transcription is underway to allow

the trancription complex to move through the nucleosomes

oPOST TRANSCRIPTION MODIFICATION AND REGULATION

RNA splicing and the removal of intronso snRNP: recognize specific sequences at the exon/intron borders called consensus

sequences (snRNPs are general molecules because of this)

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pull the exons together and cut the intron; fuse exons exons cannot change their order

o the splicesome: genearl complex of proteins that recognize the consensussequences (that spans the exon and intron); snRNP is one of those proteins

oo also does alternative splicing but determined by regulatory proteinso **diseases associated with splicing- mutations can take place on the introns which

cause splicing machinery to not be able to recognize the introns- introns are left in a

ribosome will translate the wrong protein

alternative splicing:o the way in which introns and exons are spliced differentiates between

proteins coded from the same DNA gene

occurs in the nucleuso tropomyosin: muscle protein which may participate in intracellular

transport in nonmuscle cells-- > tissue specific

o modular structure of proteins allows it to do thisjust leaves out certainmodules

o not every protein does thiso diseases can occur this way- inferface with splicingcauses a frameshift of

sorts

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o number of actual functional proteins is very small the the potential numberof mutations

Before the RNA eaves the nucleus a Gcap to the 5 end and a poly A tailto the 3 end areadded to increase the stability of the mRNA molecule

o Gcap-guanines help stabilize the RNA- protects it from RNases; helps the mRNAbind to the ribosome

o Poly A tail: stabilized the mRNA; acts as a signal that mRNA has been throughprocessing assists with nuclear export

First there is a AAUAAA-> signal for an enzyme to cut the pre-mRNA; afterthat the poly A tail is added

o this idea of stabilization is familiar to the telomeres protect the ends of theDNA

o in prokaryotes the ribosome just comes and sits on the 5 end where the AUG startcodon is; but in eukaryotes the ribosome will assemble at the

Gene families: group of closely related genesone gene undergoing separate mutationso one gene is the original gene ex. Myglobino other genes in the family are mutations; if the mutated gene is useful, it will be

selected for in succeeding generations; if the mutated gene is not useful the

functional copy is still there

o mutations result from duplications- creating multiple copies of the geneo ex. The globins

oo in humans we have 3 alpha-globins and 5 beta-globins; each hemoglobin molecule

contains two identical alpha-globins and two identical beta-globins

o differential gene expression of the different types of globins Gamma-globin- found in the hemoglobin of the fetus binds more tightly to

Oxygen that adult hemoglobin- ensures that the oxygen in the placenta will

be transferred from the mothers blood into the fetus blood

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Gamma-globin might be harmful for adults- because it holds onto the oxygentoo tightly and doesnt release it when needed for cell respiration

hemoglobins with different binding abilities are present at differentstages of human development

o expression levels may differ in difference tissues sickle cell anemia: will only manifest after birth because it is in the beta-globin

o sometimes people with sickle cell or other disease will express gamma after birthwhich makes symptoms less bad

inhibitory RNA: RnA that can bind to mRNA and break it down or prevent it from bindingto the ribosome

ENVIRONMENTAL FACTORS CAN AFFECT GENE EXPRESSION

presence of regulators impacts gene expression ex. Droughtsignal for regulator to be made- then regulator goes back to the nucleus and

turns on genes needed in condition of drought

stress response element: antother examplein stress a regulatory protein is made thatbinds to the SRE to activate production of genes that you need in response to stress