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Katie Nash Bio Exam 3, Review Guide Chapter 11 CLASSIC EXPERIMENTS IN BIOLOGY Classic Experiment #1: Griffith transforms

s bacteria (1928) The central Dogma is not yet known

Griffith looks at two strains of streptococcus pneumoniae o R strain: harmless o 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 that codes 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 exchange genetic material Refinement of Experiment #1 Avery, McCarty and MacLeod (1944) Asked what macromolecule was the genetic material or the material that transformed the mouse 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 all scientists were convinced ***not sure which experiment is right

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 the genetic 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 DNA o 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 that Surfur 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 with bacteria Centrifuge so that the less dense material is the bacteria without the virus and the denser is the 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

Eukaryotic cells can also be transformed with DNA o Can be done with plasmids, small linear molecules of DNA o 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 Crick o 1.Chargraffs rules: the # of G= # of C and # of A=# of T; knew that they bonded together- base pairing

o o o o

2.Franklins X-ray crystallography: images show a helical repeat 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 4.Positive hydrogen bonding between bases talk to chemists about how bases will interact; were originally looking at the wrong representation of guanine

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 as templates? Three possibilities for how replication occurs:

o Semiconservative: one original strand is the template for one new strand o Conservative: complete replication o Dispersive: part of new and old mixed in together Tag light and heavy DNA; originally only heavy and then put light nucleotides in the mixture 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 disappearedagainst conservative Second generation: partially intermediate, partially lighteach of the strands from the first generation 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, gNTP o DNA fragment (template and primer) o DNA polymerase o Mg2+ -- masks the negative charge on the phosphate groups

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

REPLICATION Directionality of Replication:

o o o

o o

Nucleotides can only be added to the 3 end of the DNA strand Phosphate group is attached to the 5 carbon and OH group is attached to the 3 carbon ATP, GTP,TTP come in to form the phosphodiester bond with the next nucleotide in 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 process 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 This also explains why we need a primer for DNA polymerase cannot hydrolyze the phosphate bond and obtain energy needed to bond nucleotides RNA polymerase has a way to do this

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

Read nucleotides on the template from 3 to 5 Build nucleotides on complement from 5 to 3 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 starts again; as we unzip more we add more primer o DNA ligase: joins together the Okazaki fragments; while DNA polymerase I replaces the primers with DNA Summary of Events: Prokaryotic Replication o 1. Helicase unwinds the double helix using energy from ATP o 2. Single Stranded binding proteins stabilize the unwound helix o 3. Primase: makes RNA primers on both the leading and laggings strands o 4. On the leading strand DNA is synthesized continuously 5 to 3 towards the replication fork by DNA polymerase III o 5. On the lagging strand DNA polymerase III elongates Okazaki fragments from the RNA 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 a exonuclease: can remove nucleotides o 7. DNA ligase: bonds the space left between what used to be an RNA primer and the end of the adjacent strand; requires ATP o o o

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

Read nucleotides on the template from 3 to 5 Build nucleotides on complement from 5 to 3 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 starts again; as we unzip more we add more primer o DNA ligase: joins together the Okazaki fragments; while DNA polymerase I replaces the primers with DNA In Eukaryotic Replication, Multiple Replication forks speed up the process: ligase connects the pieces o o o

Eukaryotes have proof reading mechanisms that prokaryotes dont haveprokaryotes dont 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 off o DNA polymerase III has proofreading mechanism recognizes changes in widthshould 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 DNA o DNA polymerase I adds the correct bases back in repair mechanism removes abnormal bases that have formed because of chemical damage and replaces them with functional bases o Deal with damage from chemical reactions that can damage DNA o Excision repair proteins: excise the damaged base and some adjacent bases o DNA polymerase I adds the correct bases by 5-3 replication of the short strand


Telomeres: the ends of chromosomes (telos in greek- end; meros- part) the ends cannot be left raw because 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 guaninescan make 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 replication o 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 stranded o leads to Telomere attrition: shortening of telomeres after series of replicationsafter a while the cell di

telomerase: enzyme that replicates telomeres or chromosome ends contains a piece of RNA that acts as a primer o most cell types do not express telomerase the symptoms of aging have been connected with the shortening of the chromosomes o but turning on telomerase in the wrong cells makes rapidly dividing cells immune to 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 immortality o telomerase is an interesting target for cancer drugs

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 the central dogma Archibold Garrod a physician in London who had several patients who suffered from black urine disease; the disease appeared to be inherited through families called it inborn errors of metabolism o 1909 he proposes that the disease resulted from the inability to make a certain enzyme something should be metabolized before it gets to the urine, but it is not in this case o accumulation is problematic Classic 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 media do this many times to produce varying mutations identified mutants that can survive on a complete media (media supplemented with all 20 amino 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 media without 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 add argenine (grew a sample on minimal medias each with a different amino acids) found three different genes mutations that required the addition of exogenous argenine

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

the prokaryotic central dogma o no nucleus in contrast to eukaryotes where transcription occurs in the nucleus and translation occurs in the cytoplasm or on the RER;

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 do not 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 transcription machinery 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 ori no 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 creating an 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 of pyrophosphate molecules Termination: 1. a particular base sequence signals to terminate transcription 2. DNA polymerase separates from the template o Comparing Transcription and Replication Replication How nucleotides are added To the 3 end using energy from the phosphate bond to create the phosphodiester bond How the template is read From 3 to 5 Enzymes involved DNA polymerase III, DNA poly I, II, ligase, helicase, primase How much template is used Entire genome Which nucleic acid is made DNA transcription same

same RNA polymerase Small portion of the genome RNA

Is a primer required How do we know where to start? How do w know when we reach the end of the process

Yes Origin End of the genome ter

No Promoter Termination site

TRANSLATION the genetic code: with 3 letter words there are 64 possible codons o triplets o this means that the code is redundant but not ambiguous third base in a codon is often not specificwobble- not picky in the third position; if we are picky it is between purines and pyrimidines silent mutations are often at the 3rd nucleotide- wont make a difference in translation o contains stop and start codons o nearly universal across species o explains how a single nucleotide change can result in the single amino acid substitution

o o 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

the stem like 3D structure of the tRNA molecule allows it to bond to both an amino acid and the mRMA transcript as well as have a unique structure o this structure is formed by hydrogen bonding between tRNA base pairs o also hydrogen bonds with mRNA o has an anticodon that corresponds to a particular codon on the mRNA o 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 synthase o 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

o o the synthase reads the anticodon and adds the right amino acid The ribosome: made up of RNA and protein rRNA (there is also mRNA, tRNA, mtRNA) o Large and small subunits o 3 binding sites or pockets in the large subunit in which the tRNA will bond o 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 not in prokaryotes o 2. Metionine charge tRNA binds to the AUG start codon to complete the initiation complex (tRNA + small sub unit + mRNA) o 3. Large ribosomal sub unit joins the initiation complex with methionine charged tRNA occupying the P site o initiation factors: proteins that put mRNA, ribosomal subunits, and metionine charged tRNA together o there will be an overhang of info before the start codon that does not get translated

Elongation o 1. Charged tRNA whos anticodon is complementary to the 2nd codon binds to this codon at the A site o 2. The large sub unit catalyzes two reactions demonstrating peptidyl transferase activity a. breaks bond between the tRNA in the P site and the amino acid on that tRNA b. forms a peptide bond between the two amino acidschain grows on the tRNA in the A site **the carboxyl of methionine joins the amino groups of the next amino acid o elongation factors: proteins that assist in elongation o 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 codon o ** elongation uses GTP instead of ATP not really important why o protein folding commences right away Termination: o 1. Release factor binds to the A site when the stop codon comes up o 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- so multiple ribosomes can be translated at a time while the mRNA is still be transcribed one mRNA can make many proteins not true in eukaryotes

Initiation signaled by: Termination signaled by:

Transcription Promoter sequence in the DNA Terminator sequence in DNA

Translation Start codon in mRNA 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 ribosome o 2. Go to the RER im for export or a membrane protein o factors in the cytosol recognize these signals on the amino acids the SRP: signal recognition particle binds to the signal sequence and the ribosome slowing down translation o if the signal says to go to the ER the SRP takes the ribosome to ER by binding to a signal 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 signal sequence to the channel opens the channel o the SRP then leaves the ribosome and the translation process resumes through the channel o protein is threaded through the lumen of the ER or is wrapped around the membrane 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 organelle o SPC signal peptidase complex removes the signal sequences from the peptide

POST-TRANSLATIONAL MODIFICATIONS Proteolysis Glycoylation Phosphorylation MUTATIONS Mistakes can be made during transcription but they are less serious because the mistake is only 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 this mutation; odds of having functionality is unlikely Frameshift mutation: messes up the reading framemay lead to a missense or nonsense like 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 base so that it will bond incorrectly

Chapter 13 VIRUSES What is a virus? o A virus is a delivery system for the viral genome o Viruses can only replicate after they successfully infect a host cell o The outer part of the virus is packaging for the viral genome o 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 vary bacteriophage; 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

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 Cycle o Obligate intracellular parasites: which means they only reproduce inside a host o 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 progeny viruses Animal Viruses can be DNA, RNA, single stranded, double stranded; may or may not have a lipid layer Enveloped viruses: derive lipid layer form the plasma membrane; o note that underneath the envelop is the normal capsid layerprotein o the nucleic acid has to get out of two layers o 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 the virus because we dont have that (only DNA dependent) 4. mRNA transcripts are translated into viral proteins capid proteisn, glycoproteins for membrane, etc. o some viruses have early genes and late genes: early genes are transcribed to rpoduce essential proteins for the remainder of transcription and translation

5. New virion is constructed and it fuses with plasma membrane of host to be release from the 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 release capside and viral genome 3. Transcription and translation of viral proteins and construction of new virus to be released 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 adaptation why we need a cocktail of drugs for HIVit has adapted antiviral drugs o have tried to block virus from entering the cellcould not do it o can inhibit RNA dependent RNA polymerases- needed by the virus but not by the cell used in HIV drugs o also target reverse transcriptase HIV o 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 with reverse 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

6.Viral DNA is integrated into the nuclear DNA, known as a provirus (what is integrated into 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 as opposed to in pieces polyprotein is made and in order to be active needs to be cleaved by viral protease 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 genetic material between each other Conjugation: bacteria sex o 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 material o F+ can form sex pili and F- cannot o 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

Transformation: o Observation: Griffiths experiment with the mouse o Conclusion: bacteria can take up bits of genetic material from their environment o 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 and host DNAsometimes host DNA gets inside o when that virion infects a new cell it will have old bacterias DNA o this process is important to bacterial resistance to antibioticstransform the resistance factor plasmids: small circular chromosomes that are present in addition to the main chromosome o convenient way to deliver genes to bacteria for research

bacteria can exchange plasmids during replication, some plasmids contain antibiotic resistant genes

GENE REGULATION IN PROKARYOTES affinity of the promoter sequence: In prokaryotes the affinity of the promoter for the RNA polymerase 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 but the 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 the promoter that also stimulate or repress transcription o The presence and availability of these proteins is what regulates transcription in eukaryotes An inducible system: requires an external signal to induce the gene expression; not always on

Ex. Lactose breakdown When lactose is absent a repressor regulatory molecule is bound to the DNA blocking RNA polymerase o In the absence of lactose, the repressor is boundthis is its default state o 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 be translated A repressible system o ex. Trp Operon gene for the production of TRP o 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 is activeno 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 o o

Mutation in a specific gene- mutant enzyme is made


**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 the nucleus 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

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 translation o Also needs more proteins for structure- compartmentalization Multicellular eukaryotes

Genes for transcription control tells each cell how to differentiate- turns genes on and off RNA processingcreating tissue specific mRNA The tissue formation and cell-cell singaling is not relevant in unicellular organisms CENTRAL DOGMA FOR EUKARYOTES

Eukaryotic transcription o Both eukaryotic and prokaryotic genes include promoters o The terminator sequence signals the end of transcription *** it is not transcribed Introns and exons: eukaryotic phenomenono the pre-mRNA is the unprocessed mRNA which is not yet ready to be translated

nucleic acid hybridization: used to find introns o 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 to the 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-mRNA transcript


REGULATION OF TRANSCRIPTION IN EUKARYOTES initiation: presence or absence of certain transcription factors o **the operon is specific to bacteria and prokaryotes, not eukaryotes o in prokaryotes- promoter consists of recognition sequence and TATA box o in Eukaryotes: RNA polymerase II cannot bind o the promoter without transcription factors (TF) requires many before it can bind and then more before it begins to transcribe o TFIID: first transcription factor

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 different motifs o the protein side-chains all interact witht eh chemistry of bases and DNA- bind to the major grooves o helix-turn-helix development o helix-loop-helix immune system o zinc finger steroid hormone receptors o leucine zipper cell division chromatin remodeling: nuelceosomes block both initiation and elongation steps of transcription; too free the DNA a remodeling proteins binds and disaggregates the nucleosome so that the transcription complex can bind and RNA polymerase can begin transcription; a second remodeling protein binds once transcription is underway to allow the trancription complex to move through the nucleosomes o POST TRANSCRIPTION MODIFICATION AND REGULATION RNA splicing and the removal of introns o snRNP: recognize specific sequences at the exon/intron borders called consensus sequences (snRNPs are general molecules because of this)

pull the exons together and cut the intron; fuse exons exons cannot change their order the splicesome: genearl complex of proteins that recognize the consensus sequences (that spans the exon and intron); snRNP is one of those proteins

o o o

also does alternative splicing but determined by regulatory proteins **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 nucleus o 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 certain modules o not every protein does this o diseases can occur this way- inferface with splicingcauses a frameshift of sorts

number of actual functional proteins is very small the the potential number of mutations Before the RNA eaves the nucleus a Gcap to the 5 end and a poly A tail to the 3 end are added to increase the stability of the mRNA molecule o Gcap-guanines help stabilize the RNA- protects it from RNases; helps the mRNA bind to the ribosome o Poly A tail: stabilized the mRNA; acts as a signal that mRNA has been through processing assists with nuclear export First there is a AAUAAA-> signal for an enzyme to cut the pre-mRNA; after that the poly A tail is added o this idea of stabilization is familiar to the telomeres protect the ends of the DNA o in prokaryotes the ribosome just comes and sits on the 5 end where the AUG start codon is; but in eukaryotes the ribosome will assemble at the o

Gene families: group of closely related genesone gene undergoing separate mutations o one gene is the original gene ex. Myglobin o 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 gene o ex. The globins

o o o

in humans we have 3 alpha-globins and 5 beta-globins; each hemoglobin molecule contains two identical alpha-globins and two identical beta-globins 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

Gamma-globin might be harmful for adults- because it holds onto the oxygen too tightly and doesnt release it when needed for cell respiration hemoglobins with different binding abilities are present at different stages 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 birth which makes symptoms less bad inhibitory RNA: RnA that can bind to mRNA and break it down or prevent it from binding to 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 that binds to the SRE to activate production of genes that you need in response to stress