Exam 2 : bacteria, replication, transcription, translation

Base excision repair

like nucleotide exision repair but one base at a time enzymes detect a pertubration in the structure of the double helix -demanination converts a cytosin base into a uracil -the uracil is detected and removed, leaving a bseless nucelotide -the baseless nucleotide is remoced, leaving a small hole in the DNA backbone -the hole is filled with th eright base by a DNa polymerase and the gap is sealed with ligase

RNA transcirbed in both prokaryotes and eukaryotic cells only eukaryotes (5)

mRNA rRNA tRNA pre-messenger RNA small nuclear RNA small nucleolar RNA mircroRNA small interfering RNA

components required for protien synthesis in bacterial cells: intiation mRNA fMET - tRNA 30S ribsosomal subunit 50S ribsosomal subunit IFs

mRNA: carries coding instructuions fMET - tRNA: provides first amino acid in the peptide 30S ribsosomal subunit: attached to mRNA 50S ribsosomal subunit: stabilizes the tRNAs and amino acids intiaiton facots imoirtant for delivering fMET ti itiation codon, bindign GTP binding 30S subunit to mRNA, etc

difference b/w eularyptic and prokaryotic trasncriptions

mRNa must leave nucleus, much more complicated - once rna is processed it can be exported thriugh nuclear door, moves into cytoplasms to find ribsosome and can be translated. a bunch of things need to haooen, mRNA needs to stick around linger, isntead of getting translated right away, its also unstable so need modifications to make it more stable both eularyotic and prokaryoic genes include promtoters - structure diff in euks the transcriptiona starts site (TSS) is where transcription begins in euks (vs, +! for proks) the terminator is where transcirptione ends - more complicated and not ss well understood in euks there are introns and extons in euks - the pre-mrNA is the unprocessed mRNA which are not reayd to be translates

phenotypic effects of mutations: neutrla

many chnages in the protien. sequence hae little consequence. the two AAs may have similar properties or the part of the protien might be able to tolerate changed only a small% of enzyme is the active site, where the chemistry is really interacting with the substrates --> maybe identity of all AA's all around arent very important usually AAs on outside of protien are polar - maybe chnage from polar to non-polar may see a lot mroe effects bc no polar doesnt want to be in contact with solvent - could cause lots of issues

how long does mRNA last

not long in proakryote s, RNA isnt generally stable, 5' end willprob start being eaten by exonucleases and cant be translated anymire. process stops until e transcribe the RNA next time --> maybe minutes longer in euks bc have protectvie cap and tail but maybe hours --> so to make more of tht PROTEIN WE NEEd to go back to trasncription. another singla myst come to go back and trasncribe and make new mRNA protiens lasta. little longer thna mRNA becofr beign borken down and recycle dinto component parts

F' cell HFr

a cell that has a f factor and also has picked up some bacterial genes, in some cases the f factor and plasmid include genes that were missing in the recipient cell (experiment) its very common for plasmids to have genes from the bacterium the plasmids fuse with the genetic chromsosme and then can leave, so the plasmid becomes part of the genome and then leavbes and comes back, sometime it takes some genes from the bacterial genome with it HFr - when f-factor is In the chromosome rather than separate on the plasmid

formation of a peptide bond b/w amino acids explain mechaismsm and whta kind of rxn this is

a dehydraiton rxn: oxygen from the carboxylic acid group interacts with some hydrogens from the amino agroup --> lose a water and a bond forms between the Carbon from carboxylic acid and the nitrogen form the amino rigid, defined chemistry. protiens also have directionality, so amino group is always at the front (amino terminus - N terminus) back is the hydroxy-terminus, or C-terminus

Transduction

a genetic exchange mediated by bacterial viruses viruses that infect bacteria are called bacteriaphage: virus is a way to trasnfer genetic material to a cell, it delivers genetic material that codes for the virus, introduces viral nucelic acid into the cell and makes the cell make more viruses for it---> hostile takeover! phage are distinct from the kinds of viruses that infect eukaryotic cells - good for genetic studies bc dont have to worry about getting sick

elongation of translation in prokaryotes: part 2 describe process of forming a peptide bond b/w the firts and second amino acid and what happens to theyre placements

a peptide bond forms b/w the amino acids in the P and A sites, and the tRNA in the P site releases its amino acid creating a dipeptide tRNA without AA is uncharged (already used tRNA) so peptide bond beign formed but p is also transferring to A this step also requires elongation factors and GPP

splicing

a post transcirpitonal modification whereby introns are removed, some exons may be removed (altternativ esplicing) only exons code for protein and can end up in mRNA. introns are intervening sequence, piece of rna that are made that should never be made into protien. just breaks in berween exons. boht introns and exons are transcribed splicing - we keep all econs ribsosome doesnt know whats an intron so if left in ribsosome will make the wrong proteins write at the border theres consensus sequences that where we're gonna cut them

Addition on Poly-A taisl and G-caps

a post transcirptional modificaiton - occurs in nucleus bulky methyl 7-mG cap added to 5' end and a 3' poly A tail madeof whole series of A's added to 3' end which both increase stability of teh mRNA molecule and are essential for export from the nucleus and interaction with the ribososme ( also signal to ribossome to start translating - signla to nuclear pore that it should leave)

The nucelotides and naming why is dna negatively charged

nucleotide: base, sugar, phosphate group : base + sugar, phosphate group may be missing every nucleotide has at least 1 phosphate, in otder tog et nucleotide attached to DNA, must have 3 phosphate groups

similarities between transcripiton and replicaiton

nucleotides added to 3' end wjere energy comes from for anti entropy step of adding new newcleotides to growinf chain, cleaving two phosphate groups reading and synthesizing in th esame direction using pilynmerase in both (DNA vs RNA)

how is tRNA an adaptor strucutre of tRNA visual - how anticodon is read

tRNa reads the mRNA by formign complementary base pairs via the anticodon tRNa also bring th eapporporiate amino acid to the party vis th e3' enf trNA are constantly donating the right animo acid to the protien mRNA is always read from the 5' end

transcirption onyl takes place on one strand at a time: RNA made in a 5 to 3' direction

temmplate strand and non template strand for any speicifc gene only one strans is the right strand, so still gonna forma. bubble and open up DNA to get access to the code to start making RNA, still adding new nucleotides onto 3' end ony need RNA polymerase which doesnt require a primeer, just sits down on DNA and starts making RNA. also can act as helicase pulling strands apart, melting DNA, and its gonna know which strand is the roght strand and is gonna start polymerizing RNA unitl it comes to a stop signal, a temrinator sequence, and then it will fall off --> so its only gonna copy the one piece of RNA as we're making the RNa the first part is already coming off the DNA. w edont want it to stay complexed bc we need th erna to go to the ribososme to be translated, the front end (5') is coming away from the DNA while we're still adding nucleotides ontot he 3' end till we get to the end of the gene

triphosphates

the "Active" form and incorporated into RNA or DNA examples: dNDP - deoxynucleoside siphosphate nucleoside triphosphate (NTP) - Adenosine triphosphate (ATP) ATP is our cellualr energu made in mitochindria, its a ncyelotidem use this molecule to make RNA, has a OH at 2' end presence of high energy bond - triphosphate is essential during replication

how are tRNAs charged witht he appropriate amino acid, espeically if tRNAs are constantly donating AAs, there must be a way for them to be regenerates

the Aminoacyl - tRNA synthetase enzyme charges the tRNA witht he approproate AA by reading the anticodon different enzymes add different AAs has a special bindign site for AA. reuqires ATP, Also binding site for tRNA. speical way anticodon is read. diff tRNA synthetases for diff AA. this one binds AA "a" and wil only bind tRNAs that should have that amino acid added tot hem - so theres diff amino synthetases that read th ediff AAs

what are amino acids what composes their structure

the building blocks of protein 1. An H- hydrogen group 2. An Amino group +H3N 3. a Carboxyl group COO- (acididc part) 4. R - radical gorup (side cahin) - this is what gives amino acids their chemisty. changes chemistry from simplest form H to most complex form as a two ringed structure

Primers in replication: leading and lagging strand (and visual) what happens when replication is complwhat happens when replication is completeete

the leading strand only needs one primer form ehivh DNA is added lagging: dna polymerase comes in until it hits primer ahead of it, stops when it hits the form. and primase comes in to make another piece of DNA to build off of - process is discontinous - creates okazaki fragments

mutations: subsitution

changing one letter - could lead to different maino acid being coded for in a protein, especially if one of first two locations because of wobble may be in coding, noncoding, or regulatoru regions least of a problem cause because only effects a single codon in a protien one change = point mutation - an insertion, deletion, or change of one base

what catylizes the formation of peptide bonds how does the ribsosome know what order to put the amino acids together in?

the ribsosome the mMRNA mRNa binds to ribsosome and ribsosome uses that info to figure out which amino acids to put in whay prder. once theyre alighned we;re gonna forma. peptide bond b/w amino acids and then read the next codon of mRNa and put it as the next amino acid

Transformation specifics: competency? Davis u tube? procedure speficics?

has to be homolgy b/w the DNA and some part of the genome of the bacteria so there can be a crossover event - some similarity that encourages bacteria to swap its own sequence for an extraneous piece of dna extraneous dna on one side bacteria on other; dna would be able to go through pores bc little biece sof dna are much smaller than bacteria -> able to see a transformation --> direct physical contact not requires heat shock to 50 C for short time. add divalent cations Ca+- some effects on plasma membrane to make it more porous to allow negatively charged dna in. membrane itself is also negatvieley charhes, like phospjate groups, so wihtout the calcium present the DNA doesnt wanna give up close to theose phosphate groups, so calcium shields the negtaive charge

Pentose sugars

have 5 carbons, each has an oxygen, or water aassociated with it (carbohydrate) main different is RNA, ribose has an OH attached to 2' carbon deoxyribose: pentose sugar is deoxied at 2' carbon, can tell just by looking at 2' carbon

PCR overview

have primer thats ~20 nucleotides long and complementary in sequence to target DNA taq ppolyemrase from hot springs gonna do 20-35 repeating cycles cycle ebegins by heating rxn mixture to 95C to denature DNA brekaing H-bonds that hold strands together Then redcue temp to 60 degrees so primers can form H-bonds, or anneal to complementary sequences in target DNA (too much kinetic energy at higher tmeps) temp raised back to 72C bc thats where taq polymerase fucntions optimally, begins polymerization, adding nucleotides to the 3' end of each primer attached to the dna strand after 1 complete copy, there are 2x strand copies of target DNA after cycle 2: 4 copies of target DNA after cycle 3: 8 copie sof target dna omnly 2 of the double stranded copie consist of jist the target fragment. other include flagging regions

Lederberg and tatum experiment: genetic exchange

hwo do we know that genetic exchange happens between bacteria made autotroph strains that are missing one ingredient, each cant synthesis one thing and so neither strain can grow in minimla media but when theyre mixed, some colonies grew, because genetic recombination had taken place so bacteria can synthesise all necessary nutrients (genetic exchnage and recombination took plac eb/w 2 mutatnt strains) 1 wasnt enough to convinvce ppl of genetic transfer so made auxotrophs lacking two thing ppl werent impresses and after they were able to survive missing 3 things people believed it

wobble hypothesis

idea that third position in codon is not important at all or only important if its a purine or a pyrimindine. when we're reading 3rd position in translation with tRNA the way it lines up is a lot less stringent --> a weaker interaction

features of the genetic code and codons (6)

- triplet -alomst universal - most organisms use the same code with little exceptions -degenerate - same amino acid can be coded for by multiple cododns -unambiguous -non-overlapping -61 sense codons and 3 stop codons (non-sense) - when we incorportaye a stop codon where it dosesnt belong its a non-sense mutation -initiation codon is a sense codon

somatic mutation

A mutation that occurs in the body cells. Cannot be inherited. mitosis creates population of mutant cells ex. skin cancer, mutation in skin cells than wont be passes on. may also have no phenotypic efef

phenotypci effects of mutations: gain of function

if disease is inherited as dominant, in heterozygous state, you have disease dominant allele might have gain of function causing something to build up OR it could be that th e50% of the normal enzyme activity isnt sufficient sometimes gain of function effects timing of events during development: someone born with effects mutation may have alreadt played out bc lot sof developmental things happening in utero. - we may only be seeing the end of the process

whats the 30S intiation complex>

int rasnaliton of prokaryotes, it includes th esmall subunit of the ribososome, the mRNA, tRNA fnet, intiiation factors, and GTP

arthur kornberg - nobel prise 1959 what was missing

isolated the first enzyme capable or polymeriing DNA in a test tube (in vitro) enzyme was DNA polymerase 1 - can only add nucelotides onto EXISTING 3' end what was misisn eas a short piece of primer that could act as a template for dna polymerase able to create duplex so poylmerase was able to use it to make DNA

What is a GMO

its NOT controlled mating in ordee rto get bettwe domesticated crops/animals we're adding a gene to another organissms - introduciton of new gene - only can make incremental changes at best can produce drugs in bacteria

The F factor and what does it include

its a plasmid that directs conjugation an F+ cell has the f factor on a plasmid and F- cell has the F factor on a plasmid and some bacterial genes on the plasmid as well includes origin of replication, genes that regulate plasmid transfer to other cells, and sequesces that regulate insertion into the bacterial chromsome All the things needed to be F+: genes for how to make sex pilis, how to send genetif info through tube, etc.

what is telomerase, why do we need it? process

its an enzyme whose specific function is to replicate telomeres (chromosomes ends) the telomere has a protruding end with a G rich seuqnec, first it must be untucked telomerase enzyme carries with it a piece of RNA to act as a primer/template; the RNA pat of teh telomerase is complementary to the G-rich strand and pairs with it, providing a template for the synthesis of copies of the repeats telomerase adds onto the long end of the strand, bc its the 3' end and acts like DNA polymerase, using DNA nucleotides to add on. then the template slides donwso we can make more. telomerase eventually falls off. the short end is still not extended. Now primase and DNA polymerase 3 come in and elongate the short strand overall: telomerase extends the DNA, filling in the gap due to the removal of the RNA primer when telomerases is active we dont get shortenign of the telomeres but its turned off when we're not growing anymore and its not active in somatic cells

What does DNA polymerase do? overview

its an enzyme: a polymerizer - make a polymer of nucleotides, which is DNA making nucleotides with a lot of entrop and disorder have order, so you need an enzyme to help get over the decrease in entropy

how does RNA look in transcriptions theory about RNA and reasons

its the least tructured; long string of nucleotides that leaves the nucleus and goes to the ribososmes to be translated - pictured as streched out piece of nuclei acid probably first macromoelcule to exsits - able to serve as both a genetic material (like DNA) and and enzyme/rybozyme (like protiens) (an enyzme made of RNA - sometmes telomerase called RNA enzyme bc carried RNa moelcule) Ribozymes are also self-replicating - make copies of themelves in constrast to DNA that needs a whole host of speoial proteins and conditions. believed they evolved first, they can do BOTH things dna can do, cause it has the same code so it can potentially serve as the genetic code, as it does in many viruses today protiens act as catylysts and RNA can act as them too in some sitiation

rho dependent temrination

stem loop still forms but not sufficient to destabilize the complex. need a protein called rho which has helicase activtirid rho binds to an unstructutred region of the RNA and moves toeards its 3' end when RNA polymerase encounters a terminator sequence, it pauses...and rho catches up using helciase activities , rho unwinds the DNA/RNA hybrid and brings transcription to an endp

Euchromatin

undergoes normal cycle of condensing and decondensing - condensed in M phase but decondensed in other parts of cell cycle to allow for replication anf transcription most DNA in the cell fit this description

advantages of bacteria and viruses for genetic studies

- reporduction is rapid -many progeny produced -haploid genome allows all mutatinos to be expressed directly -asexsual reproduction simplifies the isolation of genetically pure strains -some bacteria can be gorwn in the lab and require little space -genome are small (much smaller thna euks) -techniqwues available for isoalting and manipulating their genes -have medical importance -can be genetically engineered to produce substances of commercial value (ex. insulin)

Features of DNA polynucleotide structure as described by watson and crick (DNA picture)

1. H-bonding pattern b/w A-T (2) and C-G (3) 2. sugar phosphate backbone on exterior 3. antiparallel strands - nucleotides attached together via one sugar to the next phosphate groups = backbone -bases: perfect bond length of hydrogen bonds - not too close so repel not too far apart so weak, knew how long length had to be -GC makes 50% mroe hydrogen bonds than AT base pairs - additive effect over long range -antiparallelt, point in opposite directions publsihed 1953, nobel prize 1962

how phage cells infect bacterial cells: phage lytic T4 cycle

1. Phage is absoarbed to bacterial host cell 2. phage DNA is injected; sends signals to break down the host cell chromsomes (break up abcterial DNA = degraded) 3. Phage DNa is replciated, goes through transcription/translation - phage protein components are synthesized - more copies of phage genome - gets cell to make all parts of phage to make new phage 4. Step 4: mature phages are assembled 5. host cell is lysed. when cell gets too gull it bursts open - phage are released

3 categories of chemical changes in DNA

1. Spontaneous changes chemically induced changes radiation induced changes

two common ways bateria are grown in the labortatory

1. liquid medium: take sugar, liquid, coloring and food and bacteria eill gorw in liwuid- can make in huge fermenters 2. gorwing on plate - put bacteria on plate with food and swirl around so it grows dilute solution of bacterial cells, put on plate with pipete in a growth medium which is suspended in gelatin-like agar. add dilute solution of bacteria to petri plate. spread soltuion evenly wiht glass rod . after incubation for 1-2 days, bacteria multiply, forming visible colonies

Ingredient for replication (4)

1. nucleotides - ACTG (building blocks) triphosphate - active form of the nucleotide 2. Enzymes - DNA polymerase 3. Template DNA: dna polymerase doesnt just polymerize dna out of nowhere - DNa fragment and primer 4. Divalent Cations Mg2+ - all phosphate gorups are negatively charged - problem with nucleuotides or dna - need cations to neutralize some of the negatvie charge, bc two negative things doent end up next to eachother

primary structure of protein

1. primary structure of a protein is its amino acid structure, order of amino acis

what senses mutations and how are they repaired (3)

1.during synthesis: theres proofreading function of polymerases, as theyre addiing th enucleotides theyre error checking and therese and othe rpolymerases present can fix mistakes they find 2. following synthesis: mismatched bases fixed by mismatch repair 3. in the case of DNa damage from UV light, radiation, mutatens - direct reversal (added group is removed) -Nucleotide and Base extinction repair DS and SS break repair bobarding cells with uv light or radiation causes a bunch of things - stops cell cycle and intiaties "emergency repsonse"

nucelosome

200 base pairs of DNA wraooed around the histone octamer (made of 8 proteins) nucleosome includes histone octomer AND DNA wrapped around it --> its considered a repeating unit

how many DNA polymerases are there in prokaryotes? which are we concerned with

5 prokaryotic DNA polymerases only concerned with 1 - removed RNA primers and replaces them with DNA and 111 elongates a new nucleotide strand form the 3' OH group provided by the primer

nucleotide excision repair

A repair system that removes and then correctly replaces a damaged segment of DNA using the undamaged strand as a guide. UV raditation produces a thymine dimer once dimer has been detected the surrounding DNA is opened to form a. bubble enzymes cut the damaged region out of the bubble a DNA polymerase replaces the excised DNA, and ligase seals the backbone --> enzyme detect a perturbation in the structure of the double helix - brings enzymes over to cleave out a whole stretch of dna, repair it, and put back in th eproper bases

What codon does translation always start with?

AUG - start codon, alwats causes one speicif amino acid to be incorporated into protein, which is methionine --> sometiems cleaved off after so dont always find it

Telomerase

An enzyme that catalyzes the lengthening of telomeres in eukaryotic germ cells.

relating the christmas tree pic to transciption

As soon as one RNa polymerase moves out int he beginning of the gene, the 2nd comes anf third -> once we intiiate trasncirption of a gene we should expect many RNAs one after another

structure of phage

Head: casahedral, protien on outside, nucleic acid (DNA) on inside, so the whole phage genome is packe dinto the head Rest is a syringe, tube with tails that allow phage to attach to bacteria that its gonna infect. --> syringe act to inject dna into bacteria

RNA polymerase in transcription is RNA polymerase in replciation is in telomeres

DNA dependent RNA polymerase: directs synthesis of RNA from DNA template (one in prokaryotes, 3 in eularyotes) DNA dependent DNA polymerase uses RNa template to make DNA nucelic acids: RNA dependent DNA polymerase

What does DNA polymerase need to start adding nucleotides during replication in eukaryotes

DNA polymerase always need a primer wheras RNa polymerase doesnt need a primer So primase (RNA polymerase) comes in and using ribonucleotides creates little pieces of RNA that act as primers and one theyre made the DNA poylmerase can come in and start adsing DNA to the end of the RNA primers

what are consenssus sequences

DNA sequences that are similar but not identitial. Two occur in the prokaryotic promtoer the TATAAT or Pribnow box (TATA in eukaryotes) this is where DNA is intiailly separated and TTGACA - or -35 region more varibaility in -35

Experiment: how did we know bacteria have to be in physical contact?

Davis U tube: two auxotroph strains were separated by a filter that bacteria could not path throguh but anything smaller thats dissolved in media could pass back and frother no prototrophs were producted - concluded that for conjugation you need physicalcontact, which makes it disitnct from other mechansisms requries physical constatc b/w two bacteria exchanigng info

conjugation, transfer of f-factor mechanisms

F+ cell, donor, piece of plasmid unravels and travels through sex pilis as a single strand to the other cell bc it seasy to build another strand on top, you generally transfer the whole thing so both cells become F+ starts form 5' end

How DNA poymerase enzyme works - what does it act as

It works as a catylyst, speed sup kinetics of reaction by lowering activation energy. created envio that makes the reaciton more favorable chemical envio around rxn we need two phosphate groups bc we get energy from breaking the bond, to make a new bond nucleotide in aqueous envio making bonds with water and gives up bonds to make bonds with dna so enthalpy change isnt big but goes from free nucleotide to stuck in polymer, giving up a lot of entropy. making bonds lowers the entrioy so we need to break the bond After nucelotide gets added and forms hydrogen bodns with complemetnary pair, then polymerase breaks bond b/w1st and 2nd phosphate

thoughts about DNA before 1940s -1910 vs 1940

LEvene observed 4 nucleotides present in approx equal amounts in 1910. revised by chargaff in 1940s by looking more carefully chemiscal composition were known, 4 nucleotides in DNA, ACTG were known, lots of amino acids in proteins ~20 but though how could just 4 nucleotides code for th ecompleixity of life

bacterial population growth curve. - 3 phases

Lag phase: right when we put cells in rich envio with resources we dont see them divide immediately; when they prepare to divide Log phase: exponential growht: more and more bacteria present. doubling time as little as 20 minutes. want this phase when doing expeirments Stationary phase: occurs wehn resource sbecome limited, exhaust media. make a lot of wast eproducts, which make it not longer a favorable envio for bacteria to grow in

initiation code and direction and anticodon + direction

MRNA: 5'.---AUG---3' anticod: 3' ---UAC ---5'

there are 61 sense codons of mRNA and 3 nonsense codons, are there the same number of tRNAs

No there are less than 61 tRNAS be of wobble --> one tRNA may be able to recognize all four of the triplets in the third position

how do bacteria evolve antibiotic resistance?

R plasmids are F plasmids that contain genes for drug resistance microbes come in contct with one another and might exchange these genes via conjugation (or other methods) often happens in clinical settings, they cna be different kidns of bacteria it doesnt matter for conjugation and can start to trasnfer R factor u can go in wiht an infection treatable with antibiotics but your bacteria meet with other antibiotic resitant bacteria and oyou end up with antibiotic resistanc egenes and end up with antibiotic resistant infection The Resistance Transfer Factor (RTF) promotes conjugation

ingredients for replication (7) vs PCR

Replication - RNA primers made by primase -DNA polymerase 3 -Divalent cations mg2+ -DNA pol 1 -gyrase (topoisomerase in eukaryotes) -helicase -deoxynucleotide triphosphates (dNTPs) PCR - DNA primers - Taq polymerase - thermo aquatics -template DNA -We need to make single stranded dna from double stranded by melting and breaking the H-bonds between the two strands -doesnt need enzyme, just heat up mixture to separate two long strands on DNA to keep them apart, we need an enzyme we dont have to keep adding that wont denature in the heat dNTPs

elongation of translation in prokaryotes: part 1 what are the differnt sites, how does the second new amino acid come on

TRNA is in P-site: where things take place: peptidile transferase then new tRNA arrives and we're going to cut off the AA form the first tRNA and make a carboxylic acid bond with the amino group of the next amino acid A site: acceptor site, where thing arrive. the first tRNA is never in this site bc when the ribsosome is assembeledits immediately in the p-site - all others enter A site E site is where things get ejected. where tRNa goes agyer theyve donated amino acids this process requires elongation factors and more GTP the ribososme is positioning the right amino acids int he correct orientation. everything is all lined up, inside the large subunit so its gonna favor bond formation, stabulizing the transiiton state and lowerign activation energy

transduction: -Davis u tube - homologous

We do see transduction occur wihtout physical contact b/w two types of bacteria be phage are smaller than the filter (mnay can fit in one bacteria) so the phage cna move from one side to the other if theres somethign homologosu recombination can occurso it gets incorporated into the genome - create a new combo of genetic material t hat may or may not be advantageous for bacteria

rho-independent temrination of transcription

a rho-independent temrinator contains an inverted repeat followed by a sting of apporoximately six adenine nucleotides the inverted repreats are transcribed into RNA. the string of Us of RNA causes the RNA poylmerase to pausel....and the inverted repeats in the RNA fold in on itself into a hairpin loop which destabilizes the DNA-RNA pairing this structure destabilizes the complex (bc of sterics and geometry)- this causes the RNA transcript to seperate fromt he template, terminating transcription == transcirption terminated when inverted repeats form a hairpin followed by a string of uracils. so its essentila we have this speicifc seauence at the end of the gene --> only prokarotes

Holoenzyme

a subunit in RNA polymerase that is a complex which includes the sigma factor

exonucleases , what they are and 2 times theyre improtant in euklaryotic replication

ability to chew out nucleotides when we have to remove primers, need dna polymerase when there are errors made a lot of the euakryotic polymerases have error checking mechanisms, we care more about getting the sequence fiht error rate even lower bc polymerases have high fidelity and cause we have all these checking mechnaisms

PCR - GMO: What 2 things do we do after w ephysically break up cell with morter, pestal, and water to get DNA out?

add chelex beads which bind divalent cations -- magnesium is a cofactor for many enzymes like dna polymerase, and nucleases so they dont chew up dna cause we assume DNAase is eevrywhere - but we dont want these beads in the pcr tubeor it will inhibt the reaction bc magnesium is necessary for PCr to work also heat up to 95 degrees C to denature DNAse and other proteins present, theyre sensistivr ehile big pieces of DNA are fine

post transcirptionla modifications - overview

all 3 only eukaryotes start with premrna that. includds introns, still deosnt have tail or cap add bulky G-Cap to 5' end. - bulky chemical blob to protect it form nucelases and ad poly5-tail to 3' end then splicing where introns are and are removes left with mature mrna thta go ot ribososme

Classic Watson and Crick experiemnt what data did they use (5)

alreayd knew the composition of DNA but not the chemical structure and arrangement of components -pauling had already odne this with one part of protienm built model for the protien alpha helix and won nobel prize - chargaffs rules: Yeast A~T and G~C keep seeing ratio -Franklins X-ray crysallography images that showed a helical repreat - x-ray diffraction -phosphate-sugar backbone must be on exterior and solvated -putative h-bonding b/w bases -inspire by paulings model building success with alpha helix of proteins original model had phosphates pointint to center of moelcule. franklin said its an aqueous moelcule andu can put neg charge on inside so changed it

DNA. synthesis by DNA polymerase overview

annealing of primer - creates a short stretch of double stranded DNA - primer = little piece of nuclei cacid that provides the 3 'OH group. then DNa polymerase enzyme comes in. SEEs A is missing, takes dNTPs (deoxyribonucleoside triphosphate) nucletide and 3'OH and makes a covalent bond, adding to the sugar-phosphate backbone

what function do telomeres serve - artifical chromsosme from yeast experiment

artifical linear chromsosmes were added to yeast cells - made some with telomere sequences on end some without . when put in yeast cell without ends got chewed up and werent able to be incorporated. when out dna with telomere ends were able to recover -telomeres protect the ends of the chormsomses form damage from enzymes whose role is to remove forgin or damaged DNA

how the amino acid is attached to the tRNA

atatched to the amino acid acceptor stem 5' -3' CCA on adeneine on 3' OH instead of H we sttach the amino acid via the carboxylix acid group - happens by an enzume when the trna comes to the ribsosome theyre charged -- > have the approproate amino acid attached to them its an ester bond

Transformation what setting does it happen in An example of an expeiremnt

bacteria take u small pieces of extracellular DNA (or plasmids) (risk of us taking up RNA or DNA form our envio is unlikely bc we have enzymes in our skin to break it down would be dna in envio if live in envip with lots of dying microbes an when they open up they spill dna into envio which doesnt get broken down immeditately so extraneious dna pieces Cells must be Competent to take up foregin DNA - cells want this when theres an envio stressor and arent able to reporduce efficiently, will be more competent and willing to take up pieces of DNA No physical contact required transform E. coli with a plasmid: have various genes on plasmids, treat abcteria to make them competent by treating with ions and calcium, get them to take up plasmids with the gene of interest to change tht ephenotype of the bactera

conjugation

bacterial sex. process is unidirectional - theres a donor cell F+ thats spiky and one of the psikes finds its way to an F- cell without an F-factir and the spike form donor becomes a tube called the sex pilis through which genetic material gets sent to the recipient the F+ cell tranfers the F factor the the F- cell to make it F+ (a donor) but all cells arent F+ by niw because it a dynamic system, bacteria get plasmids but randomly shed them

prototroph

bacterial strain that can survive on minimla media considered wildtype - can make everything they need form ions and sugar

auxotroph

bacterial strain that cant survive on minimla media considered mutant a colony that grow nly on the supplemented medium has a mutation in a gene that encodes the synthesis of an essential neutrient ec. Leu- wont grow on medium lacking leucine

chemical chnages in DNA: chemically induced changes (5) descirbe 2

base analogs: in replication, 5-bromouracil may become incorporated into DNA in place of thymine, it may mispair with guanine in next round of replication, in next replciation guanine pairs with cytosine leading to a permanent mutation but if 5-bromouracil pairs with adenine then no replciation errors occur BRU pairs with either A or G intercalating agents rings like to stack on top of one another cause stable and they stack in double heliv. other rings can slide into the stacked DNA and intercalate - push the steps apart to make room --> its. aveyr strong interaction which isnt good for DNA alkylating agents deamination hydroxyalmine

positively charged (basic) amino acids vs negatively charged (acidic) amino acids

basic: bc theyrve already been in water already acted like a base and have lost electron, gained a protton the acific negatively charged amino acids: have already lost a proton **histadine is the closes to neutral pka: not sure ifgonna find eprotonated or not, found commonly in active sight of enzymes bc of its ability to exxtract and donate a protn under the right conditions --> useful in catalyzing chemical rxns for that reason

Watson and crick suggested copying mechanism for genetic material - 1953

basically got it right - postulated idea of using template strand to make new strand u build a new strand complementary base pairs are very important. only one base will fit just right, only one size and one chemistry. correct nucleotide must be incorporated

why is mRNa always read fomt he 5' end

bc in prokaryotes espeically ribsosme recognzies it right away, doesnt have to leave nucleus all happening in side cell. as soona s5' end is availableeven if transcription isnt finished, ribsosome comes over and starts translating mRNA into a protien, even wjile polymerase is still making it reaosns why we dont need same stabilziation in prokaryotes 5' end is ready first bc its available, doesnt have to wait for whole rna to be made

components required for protien synthesis in bacterial cells: termination: release factors 1,2,3

bind to ribsosome when stop codon is reached and terminates translation

nobel prize in physiology and medicine 2009

blqckburn, grider, szostak - work they did descirbing telomeres

components required for protien synthesis in bacterial cells: binding of amino acid to tRNA: Amino acids tRNAS amioacyl-tRNA syntheases ATP

building blocks of protein deliver amino acids to the ribososmes attach amino acids to tRNAS (need ot be able to use tRNAs over and over again in translation provides energy for binding aminoa cids to tRNA

germ-line tissue mutation

can be inherited, mutant cell goes throguh sexual reproduction so you can get offspirng where all cells carry mutation and offspring where no cells caryr mutation

mutation: insertion and deletion

can cause frame shifts which shifts the whole way of reading the code and will effect every triplet read, so will code for a new protein novel protiens are never goof sent to lysosme or proteosome to break down not nicely folded proteins bc globbed protien are bad one change = point mutation - an insertion, deletion, or change of one base

chromatin structure and nucleosome H1

chromatin is a highly ordered structure with histone and non-histone protieins - allows dns to be a lot mre compacted in cell naked DNA gets wrapped around nucleosome, made up of eight histone molecules (beach ball wedges) double helix makes a double wrap around each ball Additional protein is histone H1, which acts as duct tape wrapped around beach ball nucleosome core: histone proteins and then some additional H1 proteins that hodls together the whole structure whole structure eith nucleosome and DNA wrapped around it is the chrinsisine goes through ltos of packing to get to M phase to a chromosome thats super compacted when DNA is wrapped aorund protiens its not accessible to enzymes and cant be copies or go through trasnlation/transcription without all the packing DNa could not fit in the nucleus BUT fully condensed DNA cannot be replciated or transcribed epigenetics: DNA is alternating b/w wound up and accessible states, that governs whether genes are transcribed. if epigenetic signals say it should be less wound up then its more accessible

methods through which bacteria can enhance their genetic vairability via exchange of genetic info with other bacteria

conjugation transformation transduction

Meselson and Stahl 1958 - how does cell make an exact copy of DNA - are the two original strands used as templates? 3 possibilites

conservative replication: after first round we have all strands of old DNA and all new. the original double strand is conserved completely Dispersive: take out sections here and there so always leads to a dispersive mix of old and new Semi-conservative: start with original DNA, split bonds apart, use each one to build a new strand of DNA --> used lighter and hevaier isotopes of nitrogen

Delucia and cairns experiment on DNA pol 1 - what was found

created a mutatn strain of E-coli that lacked DNA pol1 - expected bacteria could do anything or reporduct but it could the strain could repair DNA - concluded DNA pol one wasnt major enzyme invovled in replication - 1 and 2 boht have repair functions in cell lead to disocery that DNA pol 111 is the major enzyme of dna replication

spontaneous mutaiton

denovo mutation error that occurs randomly in the course of replication

The double helix and groove functionality

different nitrogenous bases will make different chemical landscapes thata re recognized by various proteins groove are functionally important: big space = major groove and smaller space is minor groove = things are mroe exposed in major groove, so when proteins come to interact with DNA they like to enter major groove bc proteins can recognize speicific dna sequences theyre supposed to bind to

RNA poymerase in transcirption

directs synthesis of RNa from DNA template (1 in prolaryotes, 3 in eukaryotes incorporates ribonucleotides instead of deoxy- doesnt require primer contains multiple subunits - complex is referred to as the holoenzyme, which includes signma factor of which there are differentn versions

What is in the PCR master mix? whats not included in it?

divent cations taq DNApolymerae dNTPs the primers template

phenotypic effects of mutations: loss of function

doesnt ave ot be zero could be a slightly impaired enzyme the fact that most diseases are inherited as recessive means u need two alleles that dont work, ex. if carrier for cystic fibrosis or MS ur probably fine

bacterial colony

each colony divides form one bacterial cell - undergoing binary finsion after binary fission calle doclony bc theyr eall different, each derived form own cella nd have diff properties than other bacteria

linear chromsomes contain telomeres

essential to preserve integrity of DNA, bc bare, unfiinished ends can be broken down by nucleases which would lead to a loss of information telomere has arepeating sequence that is not a gene, just a sequence that allows the end to be tucked in and find complementary sequence gets shorter as we age - problem with how cell divides had a G rich single-stranded overhand probably onyl about 1% of the DNA in chromsosomes codes for proteins, dark matter of denome, centromeres, telomeres - just structural parts but we dont know what rest does

differences b/w transcription and replication - how much template used

everything is replicated only transcribe each transcriptional unit starting at th promoter, start at +1 keep going until finish terminator

what happens when replication is complete

exonucleases cut out rna nuceotidies, chew out 1 by 1 rna molecules DNA polymerase 1 then takes out RNA primer and replaces is w/ DNA nucleotides because theres a 3' OH group to IMG_748711C17066-1add onto except for at the end where theres no 3' OH, this leaves a gap where that primer was taken iut -- end replication problem the nicks left after the process are fixed by DNA ligase - seals the gaps in the backbone - bonds th espace left between what used to be RNA primer and the end of the adjacent strand

Griffith's Transformation Experiment

first described the bacterial ability to transfer genetic information through a process called transformation - but didnt know it 1920: 2 strains of diplococcuse psneumoniare. 11R strain harmless but 111s strain pathogenic. heat killed 111s isnt pathogenic but heat-killed 111s mixed with live 11R IS pathogenic said 11R strain was "transformed by the what killed 111s reconstitues pathogenicicity of 111s by mixing with live 11R. What heppened: when kill 3S theres pieces of DNA in mixture (when cell is disrutped piece sof dna come off) mixes with 2R which take up pieces of DNA and some take up th eright pieces to become pathogenic 3S is smooth, 2R is roguh. smoothenss is a capsule on outside of bacteria which protect it from the immune system of the mouse so theyre pathogenic bc cant be attacked --> 2R bacteria were trasnformed - did know which cellular compoennet spit out of the heat shocked 3S caused the trasnformation --> was hard to separate DNA, RNA, etc Avery, Mcload and Mcarty extended htos work to find out what component of the heat-killed cells is causing the transformation - separated heat killed 3S and mized with three enzymes, RNAse, DNAse, and Protease, when they mized it with RNAse and Protease they still got trasnformation so knew these things werent the transformting principle but didnt when mixed with dNAse - nominated for noble prize but people stioo question how do you know the enzyme got rid of all the DNA, dodnt win

Evidence that the genetic code is non overlapping

first geneitc disease described is sickle cell anemia - one nucleotide change that causes one amino acid change -> if code were overlapping we should see up to three changes if the nucleotide was part of more than one triplet it would affect more than one amino acid

how phage transfer genetic material

font have DNA packageing mechsnism, only package by size so could take up a piece of bacterial DNA when theyre maturing and becoming assembeled (defective phage; bacterial DNA packaged can subsequently infect another cell and bacterial dna is injected instead, bacterial dna is integreate dinto recipient chromosome throuhg crossover and creation of recombinant DNA---> incoporated into genome

what gmo food are best to test

fresh genetically modified food sbc theyre dna is most in tact - frozen corn or papaya too processed or oily wont work

front end of peptide vs back end

front end is the amio group hanging off and back end is the carboxylic acid feoup attached to the tRNA

components required for protien synthesis in bacterial cells: elongation -70S intiiation complex -charged tRNAS -elongationf actors -GTP- -50S ribsosomal subunit

functional ribsosome with A,P and E sites whee pritein synthesis takes place brings amino acids to ribsosome and helps assemble them in order specidied by mRNA binds GTP and cahrged tRNA, delivers charge dtRNA to A sight, sitmilated moevment of ribsosme to nect codon, etc. provides energy created peptide bond b/w amino acid in A site and P site

what are repeat sequence mutations associated with

genetic diseases: ex. huntingtons number of repeats often increases with each generation and the more repeats correlates with more severe symtpomss so may start to see earlier onset earlier manifestation known as genetic anticipation

alternative splicing

mechanisms by which we can make multiple versions of the protien through a single gene - through process of splicing to have flexibility in th ehenome. thats why we have ~ same amoutn fo genes as other organsism but code for many more protiens than would expect. splciing allows us to break up proteins in to tehir compoenent parts, often coded in exons, so pick and choose which parts to include ina a particular rpotien allow d for multiple protiens form a single gene - we can make a version of mature mRNA with all exons or we can cleave some exons when we cleave introns, so now form one trasncript we;ve mafe two different mRNa that cna go to ribossome and will be translated to different proteins that have similar roles maybe makign a diff spliced version that better involved for the environment, or maybe has slightly differetn fucntion --> since the proteins have different structures, they also have customized functions probably NOTE: we cant change the oder of exons in the protein

Types of RNA made by transcription (4)

messenger RNA - mRNA: carries the message from the DNA to the ribososme ribososmal RNA - rRNA: a structural component of the ribsosome transfer RNA- tRNA : participates in translation, "Reading" the message and bringing the appropriate amino acid to the ribosome miRNA and siRNA (tyeps of RNAi - interfering RNA) eukaryotes only --> play ane ssential role in gene regulation in cell

minimal media

minimal diet for life - prototrophs. includes ions and a sugar source on agar plate - evertyhing bacteria needs to grow --> doesnt include AA, fatty acis, nucleotides, etc. bc they can make their own building blocks

connection of telomeres with aging why deos it get turned off?

most cells dont express telomerase the sumptoms of aging have been connected with the shortening of the chromososmes - people w dieased tleomeres have premature aging, long telomeres associated with health maybe as a protective measure bc when telomere gets short its a signal for the cell to undergo apoptosis, which is good bc it prevents cells from dividing out of control out of control cellg growth = cancer lots of times with cancel its where telomerase has been turned back on - cancer cells turn on th eexpression of telomerase as one way to confer immortality - making it a drug target for cancers its useful to have a self-destruct signal in cells that are rapidly dividing but not supposed to

comparing size of human gene size, mRNA size, and the number of introns

most human genes contain introns the range is very scatteres Dystrophoin - one of biggest genes in the genome - mRNA is much bigger. the proteins that muattaed in ppl with duchen muscular dystrophy

Polyribososmes differences in euks and pros

multiple ribossomes can be reading one mrNA concurrently (translation) so may protiens can be made simultaneouslt form the same piece of mRNA pros: mrNA is still being transcribed and ribsosome can come in and start translating becayse the 5' end is the first to come out and thats where translation strats in euks mRNa has to be processes to leave nucleus so whiele multiple risbosomes an be on the mRNA they can be happenign during trasncription - transcirption ends, additon of cap, tail, splicing, export form nucleus, and only then is mRNA available tot he ribsosome

Does the mRNa get translate by only one ribsosome?

no, in both euks and prokatyores, once th eintiation factor forms at the 5' end the ribsosme starts to move dwon and the 5' end is free again so theres now aiting, once its available another small subunti can come in and process can start over theres always going to be one mRNA translated by multiple ribsosome at any given time --> always expect more thna once protien being made from a given mRNA

nonpolar group of of the 20 amino acids

nonpolar - hydrophobic: dont form H-bonds with water, made up of a non-polar hydrocarbon, likes to interact with ohter non-polar things --> suusally found on inside of protiens bc outside is ususally exposed to aqeuous solutions. when protien is synthesized at ribososmes these hyrophobic amino acis interact with eachother right awat and form a hydrophobic core in the interior of ptoyein

chemical chnages in DNA: spontaneous changes (2 types)

occur during replication or due to spontaneous chemical changes 1. unequal crossing over during meiosis. -tetrad is mispaired at meiotic synapse - result is two unequal choromsosomes, one with a duplication, and ine with a deletion 2. deamination: cytosine losing an amine group becomes uracil --> leads to a transition mutation, specific repair mechanisms to recognize this methylcystosine loses amin group and becomes thymine --> also leads to a transniton but harder to repair because thymine is stilla. base that belongs to DNA

where telomere attrition occurs

occurs on the lagging strand on both ends of the linear chromsosme

genetic code cheat sheet what do the STOP codons mena does every organisms use this same code

onece we have the sequence in the mRNA, the ribsosome "Reads" the mRNA, translatign each codon into an amino acid means the codons dont have an amino acids nect to them, when we get ot this signal , no amino acid is incorporates, so not all codons code for amino acids yes: when robsosome reads mRNA it reads the triplet and incorporates the correpsonding amino acid or stops

both RNA steands can seve as templates for translation

only one of the two strands codes for the RNA we want. but the gene is encoded on both strands

differences between RNA and DNA (3)

oxy vs deoxy, in RNA have OH, 2' hydoxyl of the sugar instead of thymaine uracil is mase RNA doesnt forma. double helix through steric hinderance of additional OH c group so its not comfortable for molecule to adopt double strand

bacterial lawn:

phage come, infect cells and make into phage making factories last phase of -1ytic cucle is cell bursts open full of phage and all phage go to next cell. holes correspond to where bacteria used to be but bacteria was lysed and phage burst out of it hole gets bigger as phage infect other bacterium in the neighborhood

PCR-GMO: what primer did we add as our control and why

photosystem 2 - to make sure we have plant DNA - its a protein complex responsible for photosynthesis so if we see a band there we knoe we got plant dna

polar group of 20 amino acids

poalr: hydrophilic - polar groups that all hav ethe portential to hydrogen bond with eachother or with water- we wantto be in hydrophilic envios

GMO MM vs Plant MM

primer for promoter and terminal primers for photosystem 11

Replication of the bacterial chromosome

process always begins at ori - one place in circular genome where replciation always begins theres a speicific protien that recognizes it and strats to pull apart the DNA creating a replication bubble wiht two forkes that go around until meet at the bottom resulting in two doubel stranded picess of cicular DNA

The trasncitption unit: what does it include? (visual) how is it organized

promoter, RNA coding region, and terminator one deifnciiton of a gene promoter: where the major descision ahppens of whether or not gene is transcribed. where RNa polymerase intially binds and if it does it must be oriented towards the gene in order for trasncription to start, comes and reocgnizes the promoter sequence limit b/w promtoer and RNa coding region is +1. nucleotides before are reffered with negative number and are "upstream; nucleotides after are "downstream" RNA coding reigon: could be anything, tRNA for translation, rna improtant for making ribsosome, etc. temrinator: end of every RNA coding region had temrinari, need singnal for when its the end of the gene this is transcribed, becomes tail end of gene

Transclocation in prokaryotic trasnlation

rubsosome moves down the mRNA to the next codon (translaocation) with required elongation factors and GTP, this kicks the uncharged tRNa that was in the P site to the E site where it is moves to the cytoplasm Now the tRNa that occupied the A site is in the P site and now the A site is open and ready to recieve another tRNA at the end of each elongation , the amino acid that was in the A site is added to the polypeptide chain and th e A site is free to accept another tRNA

hershey-chase experiment -1952

prove DNA was genetic material grow bacteriaphage and give them radioactive amino acids for sulfer so when they build more proteins will take radioactive amino acids and incorporate into proteins - bacteriaphage grown in media with 35S, which labels protiens ( methionin and cysteine contain S) grow phase in presence of radioactive phosphorus, give them nucleotides the building blocks of dna that contain radioactive phosphorus and will incoproate into genome so have radioactive DNA - P32 labels nucleic acis -(only DNA not ptoien has phosphorus) let them infect new cells then look at newly infected bacteria and see whats inside. take out Infected bacteria (e-coli) and separate them. initially found both 35P and 32 S because held on very tightly on outside so put in belnfer. after allowing phage to attach and inject their DNA, put in blender which sheared off the protein that was just on outside of cell 32P was inside cell while 32S was not asspciated with bacteria - convincing that DNA is genetic material - must be what directs new making of phage particles won nobel prize - didnt honor assitnatn chase -1969

chemical changes in DNA: radiation induced changes (1)

pyrimidine dimers: caused by UV light which is damaging two pyrimidines, like thymine, UV light causes covalent bonds b/w the two of them, which distubs the double helix structure - doesnt allow for selection of proper base pairs in addition, ionizing radiationcan create reative ions and free radicals whihc can break phosphodiester bonds

nitrogenous bases

pyrimidine: cytosine, Uracil (RNA), and Thymine (DNA) purines: Adenine and Guanine diff b/w thymine and uracil is methyl group on thymine at carbon 5

nobel prize physiology - hep C virus

recent, important to detect in blood supply bc people were being infected from blood transfusions and long-term untreated infection leads to liver cyrosis and cancer - drug related to HIV cocktail

how does transcirption initiate (prokaryotes)

regulation of the binding of RNA pol to the promtoer dictiates the tmeporal specificity of transcirption A protien called sigma factor, part oft he holoenzyme, a subunit of dna polymerase recognizes the partiuclar 35 consensus sequence on the promotoer. if a protien binds here it wil bind and bring DNA polymerase along - dictates when gene should be transcribed (there are different signma subunits in prokaryotes that recognize different promoters. some genes will ahve the same promoters as other genes) the signma subunit acts with the -35 sequence and promoter and moves the whole RNA polymerase down so it plops onto promoter, oriented by sigma subinot. the active sight of RNA polymerase is gonna be +1 on the proper DNA strand, oritneted to start polymerizing from 5' to 3' if you want all genes tunred on at the same time; transcribe mRNA and translate proteins in concern, give them all the same sequence at 35 so theyll be reocgnized by the sigma factor and when its present, it will set off the transcription of a whole set of genes consensus seuence 10 is more downstream on the promotor and it helps get the process started, gonna aif it by onyl hvaong A's and Y's so its easier to pull apart; the intial melting of DNA

heterochromatin

remains highly condensed ex. Barr body (inactive part - 85%) except if cell has to divide it has to be accessible parts of Y chromasome, centromeres, telomeres these areas are not transcribed Exceptioion: if had to copy DNA would be able to do that

DNA helicase

replciation: enzyme that helps break H-bonds (since we cant heat things up in cell) that are holding together double helix there is one at each replciation fork

single-stranded bindign protiens

replciation:DNa is veyr sticky and wats to come back together to make H-bonds when its pulled apart so u need a mechanism to prevent them from coming back together - these clock DNA form being able to make interchain hydrogen bonds

replicaiton of plasmids

replicate independent of the bacteria chromsome double stranded DNA, have origin of replciation site (ori) - strand separates, newly synthesized DNA starts there, sepration of 2 daughter plasmids i= each have one new and one old strand bacterial cells can make many copies of plasmids but make only one copy of its circular chromsosme - easier to isolate bc theres many genes on plamsids can be transferred from to other bacteria much easier than chromsosmal dna genes that confer antibiotic resistance are often found on plasmids and trasnfer between bacterial populaiton

DNA gyrase

replication: As we open up the twisted molecule it buts more strain ont he part thats not open. This clips the DNA and puts it backtogether so we dont ahve mroe and more troubleopening the doubel helix - relaxes the supercoiling

where does the eukaryotic DNA begin unwinding and how?

replication: initiates at Ori, mediated by initiator proteins - initiator proteins bidns to origin and separates stranfs of DNA to intiiate replication

subunits of the ribsosome: prokaryotes vs eukaryotes sizes and what is S

ribsosome has two subunits prokaryotes: large subunits is 50S and small is 30s but all together add up to 70 because more compact - small subunit is 16SRNA which we compared to figure out genus of bacteria eukaryotes: larg subunit is 60S and small is 40S - adds up to 80S each subunit composed of both rRNA and protien. S is the svedberg coefficient, which measure of the size and density (rate of migration in a sucrose gradient during centrifugation)

where does the ribsosome commense translation, how can transcirption and translationt ake place concurrently

ribsosome physically recognized 5' end of the mRNA 5' end is untranslated reaction - part of mRNA tjay was transcirbed but is just before the AUG, the place where the ribsosome commenses translation, so anything ebfore isnt incorporating any amino acids translation proceeds 5' to 3', each tome it proceeds down to a new triplet, wehre its gonna incorportate a new amino acid yes, in prokaryotes, not in euks cause transcirptiont kaes place in nucleus where translation takes place in cysotol

formation of phosphodiester bond

rxn doesnt happen in a vaccum its the active sight of the enzyme - which positions reactiants to get perfect geometry. also acts on molevule pilling electrons aaway and towards, extratcomg prtoein and overall alters chemcistry around reaction sie to favor it at 3' end it can basepair with the template strand - thats how polymerase knows what nucleotide to incorporate 3 phosphate groups will be lined up in right geometry with the 3' hydorxyl to faciliate th ebond being made b/w phosphate group and 3' oH --> a nucleophilic attach, leaving group is 2 phosphate groups if wrong nucleotide is incorporated enzymes look for perterbations in the structure (wider or narrowr spaces) so a lot of dna repair mechansm

secondary structure of protein

secondary structure: itneractions b/w amino acids cause the primary structure to fold into a secondary strucutre., such asn an alpha helix componenet structure: a helix, one amino acid chain going around' stabilized by interactions in main-chain atoms - the amino and the hydroxy groups limtied # of seocndary structures building up to unlimites teritary structures

intiiaiton and elongation in prokaryotes - transcripton

signma factor is only present in cell when we want to transcribe particular genes. bidns to core rna polymerase to come down and bindt o promtoer. DNA strats melting at -10 consensus sequence of As and Ts. opens up and a bubble forms RNA pol builds a complementary strna dof rNA, unlike DNA this strna dis only temporairlty associated with the templare brings in nucleoside triphosphates and once RNA starts proceesing adding nucelotides, the sigma factor falls off, its only there to recognize th epromtoer. it cna then go interact with another molecuele of rna pol and work on the transcription fo another gene as rna pol moves down it pulls rna away forom dna and a little piece comes out like a tail int he back of the enzyme keeps making rna untilt eh temrinaotr seuqnece

types of point mutations

silent, missense, nonsense these only refer to mutations in the protein coding regions of the genome missense - codon codes for a different amino acid with the change of one base nonsense - codon now codes for a stop codon with the change of one base silent mutation - even though the coodn is changed it codes for the same amino acid --> can have anyhting in 3rd position bc all 4 code for serine - wobble

plasmids

small circular DNA molecules that replicate separately from the bacterial chromosome naturally occuring in the population - constantly gaining and shedding them bacteria may contain different types of plasmids. may be co-opted by sicientists because theyre are a useful way to introduce a gene of interest --> copying them is like heteroplasmy - its unreliable, when a cell divides unsure of which plasmids get divided up

role fo the 5' cap and poly A' tail in eukaryotic translation

small subunit comes along and binds to the 5' end but not theres a longer untralnated region at 5' and 3' ends so we wopnt find an AUG thats close to the 5' end (where in prokaryotes, small subinits come in bound to teh 5' end bind to shine delgarno sequence and boom also bound to intiator AUG) there are special prtieins that attach to the 3' poly A tail and interacr with cap-binding proteins, both of which help the large subunit come along (eventually) and enhance binding of the ribosome to the 5' end of the mRNA (so they essentually direct the binding of th e5' end of the mRNA) all these things working together to the formation of the active translational complex for making protiens, all thsi si assembeled before reaching AUG and once assembled the ribsosome can start walking down the mRNA 5' to 3; to find the first AUG, the kodak sequence thenw ill see trNA with methionine and imet come in, assembly of full ribsome , and then translation will commenc ewjere new tNRNAs keep coming in, peptide bonds and thne will contineu until reach stop codon

initiation of translation in prokaryotes

small subunit comes down at sits down at the 5' end of mRNA, ribososme binds at the Shine-Delgarno sequence, which is an essential sequence and its repsonsible for recongizing the small subunit so it needed for it to bind successfully. its also positione dover the initiation codon, AUG. then TRNA comes in with special form of methionine - fNET - formulated and other things joing to like initiation factors important for the recogniiton - and GTP comes in for cellular energy that drives rxn despite loss of entropy this is the 30S intiiation complex, which consists of th esmall subunit, mRNA, tRNA fNET, initiation factors, and GTP, only once theyre assembled does the large subiunit come in

clincial relevancy of different sizes of prokaryotic and eukaryotic ribsosomes what subunit did we use to figure out genus of microbes - whats a possibel reason we may not have found it

some antiboticis were clinicially target prokaryotic ribososmes - molecules that affetc translation at prokaryotic ribososme, - which is waht makes them effective in humans, dont hurt our cells but could disrupt our microbiome small subunit of prokaryotic ribsosome, made of 16S RNA and protien. if there was mold, which is yeast, a eukaryotic cell, we wouldnt have found the 16S RNA

differences in translation in eukarytoes (compared to prokarytoes)

the ribsosome is a diff size S and composotoion (40S compared to 30S for small subunit) mRMa mustbe transported to cytoplasm formt he nucleus. G-cap and the poly-A tail are present The g-xap and poly-A tail direct bindign to the 5' end of th emRNA mroe accessory factors are required each case has a special (but different ) intiiation tRNA - tRNA fmet for prokaryotes -tRNA imet for eukaryotes the intial AUG is found within the kozak sequence (kozac sequence includes AUG rather than AUG coming agyer the shine delagrno sequenxe (that reocngizes the subunit )

tertiary structure of protien quaternary strucutr

the secondary structure folds further into a teritary strucutr - each tube is an alpha helix -all proteins have tertiary structure -overall 3D fol of protein. two or more polypeptide chains may associate to formthis strucutre - some protiens are made of multiple subunits, or multiple polypeptide chains -only a subset of ptoeins ahve this quaternary strucute

RNA splicing -removal of introns mechanissm

there are specific consensus sequences at 5' and 3' end of the introns and they attract the sNRPs to bind there and then tehey undergo a rxn where the two bind together to form a structure. other proteins that form the spliceososme compacy and they make a clip at either end of the intron/exon border. also ahve a pasting function so the two exons are pasted together with a new phosophodiester bond exons are preserced intron structure beign broke down into diff RNA nucleotides, can jsut have new phosphate s added and recycled as new pieces of rna whats left is mature RNa that codes for proteins whoel process emdiate by big complex called th esplisosome. the snrp is onyl one componenet. we can also ahve alternative splicing to get different verisons of this protien splisosome is an endonucleas, doesnt chew from edges

how replication is coordinated on the two strands (laggin strand visual)

there are two units of DNA pol 111 , one for leading one for lagging strands and theyre linked DNA of lagging strand is looped around to create a segment of dna thats going in th esame direction as th eleading strand. When it hits the primer the loop is released and a new loop is formed

tRNA: - what does it do - components

there is hydrogen bondign b/w base pairs and the flattened cloverleaf model shows pairign b/w complementary nucleotides helps to read mRNA and brings the appropriate amino acid along with it had anticodon, comprises three base pairs and interacts with a codon in mRNA, the codon is read backwards how is the tRNa gonna tell the ribsosoem to incorporate alanine when this tRNA binds to the mRNA --> when the tRNA comes to the ribsoosme its gonna have the amino acid attached to the amio acid attachment size, always at 3' end : 5' -3' CCA

what are telomeres

they are the end of chromsosmes. the ends cant be left raw cause would be recognized by repiar mechansism and fused with other seuqeses -exonucleases, enzymes that work from the ends would recognize raw ends as foregin and chew them up its a long, repeating sequence that folds over itself at the end of each. linear chromsosme. one segemnt is single stranded and tucks itseld in. we have this same sequence over and over hwich allows the overhand to find its complementary sequence when it comes around and tucks in a lot of proteins mediate this

Dumb thing about phage

they dont have a process to know how to package their own DNA. they evoled to choose pieces of DNA based on size, not identity. Because they dont have this mechanism and they rbeak up bacterial DNA when get into the cell, occasionally they package some of the bacterial DNA instead of their own if its the right size to fit in the phage head. Goes to next cell and injects it thinking its injecting its own DNa but its a piece of bacteraial DNA when new piece gets integrated into recipient now the bacteria has new genes that come from another bacteria - the phage was justa. messenger -----> the phage can still inject the DNA into a bacterial cell because injection is controlled by the proteins in the capsid. However, because the phage DNA now contains bacteria chromosomal DNA, it no longer contains the capability of co-opting the host cell machinery, degrading the bacterial chromosome, creating new phages,, etc.

transitions and transversions affects on double helix?

transisison are purine to purine (A-->G) or pyrimidine to pyrimidine (C-->T) --> maintains width transversions are purine to pyrimidine or pyrimindine to purine --> two purine will make a bulge, two pyrimindines will make a pinch the structural anologies cna be recognized by repair mechanisms doesnt consider th eeffects on th eprotien

Meselson and Stahl 1958 - how does cell make an exact copy of DNA - are the two original strands used as templates? experiment

used two inonradioactive sotopes of nitrogen N14 and N15 which is heavier from additional atomic weight grow bacteria in heavy nitrogen so they will incorporate nitrogenous basss so all dna will be labeled with heavy nitrogen then once its incoporated intot he henome, swithc to using light nitrogen - a centirgfuge tube is filled with. a hevay salt solution and DNA fragemtns -it is then spun in a centirfugr at high speeds for several days - a density gradient develops in the tubs. Heavy DNA will mvoe towards bottom and light DNA will remain closer to the top if we have a mixture of hevay and light then there will be a band in between Results: -after one round of replication the DNa appeared as a single band at intermediate weight (ruled out conservative) -after second round of replication DNA appeared as two bandsm one light and other intermediate (was stilla mixture, but with more 14N --> a mixture of original 15N/14N and completely new 14N. we still ahve intermediate but we will have more and more 14N bc new strands act as templates --> supported template based process and semi-conservative

Is RNA single or double stranded? Whats its secondary sturcture RNA 3D strucutr

usually single many types extra 2' OH on ribose sugar causes some steric that doesnt allow RNA to adopt a doubel helix strucutre RNA folds in on itself, making intrachain H-bonds, to make a bunch of different structures rather than forming 2 chains forms stems with complementary base pairs and loops

Watson crick rosalind franklin

watson: young prodigy - question of DNA structure, sexist, racist, antisemetic, didnt contirbute much after crick: old codebreaker in war, knew what was wrong with people research, contributes a long to science after structure, credicted with central dogma both didnt do a single experiemnt took data form ppl like rosalind fraklin whose boss shared her data. won noble prize for structure but she was dead from radiation, while her boss won too

evidence for presense of introns

we figured out how long genes were and it didnt make sense if genes with 1500 nucleotides long, divided by three should be protien of 500 amino acids but only codes for protien of 200 amino acids have dna from nucleus and rna that we knew correpsonded to the protien. allowed rna to anneal ewith DNA and when both line up it forms loops, bc the dna was coding both the inton and the exon but the rna only included th eexons, dna gets loopsed out loops suggest there were sequences in the pre-RNA that werent present in the final trasncript

how is mRNA translated - overview

we have mRNA and tRNAs bindign with amino acids attached to them - there can be more than one tRNA attached to the mRNA - can be lined uo next to eachother in order now we need to get the amino acids attached ot one another ot build a protien - we need catylyst to catalyze the formation fo the reaction b/w the two amino acids in that speicfic way. theyr egonna be oriented int he ribososme process requries eneryg, need a moelcular factory for protien synthesis --> big jobs enzymes do : amino acids ar ebound next to eachother in the ribososme oritented correctly, we're already facilitated the formation of the bond b'w these two aminoa ciss --> still in ribsosome, the protein can create a whole chemical environment, ribsosome can control the chemistry of the envio, fascilaitating th eformation of the bond bond is entropically disfavored: taking two free tRNA and making amino acids come together - we're expending energy liek int ranscriptiona nf replication but instead of clipping off phosphate groups to make bonds we're gonna use cellular energy --> GTP, jsut swap out which nitrogenous base (b-list eneryg) - GTP is a very important source of energy in the cell, during translation

GMO PCR: what we're looking for promtoer we're looking for what primers did we use

we're gonna be looking for the sequence within the promoter and the sequence within the terminal, doesnt matter what gene they uces bc it will have either one CamV 35S promoter, derived from pathogenic virus, cause hteyve evolved mechanisms ot get its genes expressed at a high level - from cauliflower moasaic plant - a lot of genetically modified thigns have this promtoer we have three diff primers that have been designed to detect the sequences of the CamV promtoer, the NOS terminator, and PHotosystem 2 only 85% have the primitier and temrinal we're looking for, - cant make big conclusopons

termination in prokaryotic translaiton

when ribsosome reanslocates to a stop codon theres no tRNA with an anticodon that can paur with that codon in the A-site instead theres a protien called release factor that recognizes the codon and when it comes it int acts as a signal for the polypeptide to get cleaved off of the tRNA the RF forms a complex ith GTP and binds to the ribsosome rleease factor with GTP and other soecuak factor heko nd proces 00> polypeptide is release and the whole thing sfalls apart. the tRNA, mRNa and subunits com apart

why do telomeres pose a problem during replication?

when the RNA primer closest to the linear end is removes, DNA pol 1 cannot fill the hap, since theres no primer , 3 'OH group to add onto but there none a telomere is a non coding region but everytime the cell divides ti get shorter and shorter and at some point we run out of the non coding part into the coding part if this was all we had everytime we replicated a linear chromosome, one of the strands would get shorter. its gonna limiy how many times a cell divides when you remove the RNA primer that goes 5' to 3' you look for a place ahead (to the right) to attach DNA too but theres no place ( you cant attach it to the 3' groups