biochem exam 2

In the Griffith mouse experiment, what happened when the non pathogenic and dead pathogenic strain got mixed and injected into one mouse?

The dead pathogenic strain had transformed the phenotype of the non pathogenic strain causing the mouse to die. They found that it was nucleic acids that had this special ability to transform the phenotype of the bacteria.

what are nucleic acids by definition

chemical substances that contain genetic information.

explain the central dogma

DNA is replicated by DNA polymerase 3, a protein. Then transcribed into RNA via RNA polymerase and then translated into protein via ribosomes.

What is deoxyribonucleic acid (DNA)

the key information store of the genes that encode proteins. It is what is replicated and passed down from one generation to the next.

Ribonucleic acid (RNA) acts as..

an intermediate messenger that is transcribed from DNA and provides the templates called mRNAs for the synthesis of proteins in a process called translation. they were the first molecules and act as a catalyst that store information.

The structure of nucleic acids is key to their function as genetic information, explain why

the structure provides the energy bases for the molecule to make an exact copy of itself, can directly “ report out” when it has been damaged allowing a repair me signal to go out to the cell, and the information contained in the polymer can be accessed from the inside by seperating strands or the outside through the large grooves.

Nucleotides are what and polymers are what

nucleotides are the building blocks of DNA and polymers are how they’re put together

What is the B form of DNA

Watson crick Franklin base pairing

what are building blocks in simple terms of nucleic acids

they are polymers built from nucleotides.

what are the three parts of a nucleotide

phosphate, sugar, and base

on the 2’ carbon if the sugar, if there is an OH what sugar is it?

Ribose

What bases does DNA use vs RNA

DNA= A C G T

RNA= A C G U

what are the key positions of the sugar involved in building the nucleic acid polymer

5’ and 3’ positions.

what is a nucleoside

base and sugar, no phosphate group

what is an N-glycosidic linkage/ glycosidic bond?

the connection between the base and the sugar that occurs between the 1’ position on the sugar and a nitrogen on the base.

Nucleoside monophosphate vs nucleoside triphosphate

mono= what we see in the final nucleic acid polymer

tri= serve as high energy building blocks used by the cells to synthesize the nucleic acid polymer

how many benzene rings do pyrimidines have vs purines?

pyrimidine= 1, purine= 2

nucleotides are joined by what type of bond

phosphodiester bond with the 3’ carbon on the sugar

how do phosphodiester bonds form

3’ OH nucleophilic attack on activated 5’P. So there is base activation of 3’OH, then nucleophilic attack of 3’OH on an activated 5’ phosphate. (ACTIVATED= leaving group attached to the 5’ phosphate to drive reaction).

how can nucleic acids also be broken dow?

cleavage of phosphodiester bonds when a base gets activated by a water molecule and allows for nucleophilic attack on the bond.

why is the 5’ to 3’ orientation of long strings of nucleotides important? how doe step polymer contain information?

it matters because they have polarity and direction, and its stored in the bases.

How do nucleotides interact with each other

through hydrogen bonding to form Watson crick Franklin pairs.

how many bonds do A and T form vs c and G

A and T form 2 h bonds so they are weaker than C and G that form 3

explain the B form of the helix?

the strands are anti=paralell, sugar phosphate backbone is on the outside, nucleases are in the inside, A:T and C:G base pairs line the inside of the helix, and thene are completely orthogonal so they only pair with each other and the pairs stack on top of each other perfectly.

what are the base stacking interactions in the double helix

van der whaals interactions occur between hydrophobic nuclease faces (steric) and there is pi stacking (electron effects). they reinforce the individual base pair interactions to drive massive stabilization of double helix.

The B form DNA helix is asymmetrical, what does this mean

even when the two strands are joined together, the helix provides two grooves that provide access to nucleobases from the outside, one major groove that is wide and deep and one minor that is shallow and narrow, both showing exposed bases, and proteins bind through that major groove.

what can bind through the major groove in DNA

proteins can bind to the dsDNA

explain how the structure of sugar affords more conformational complexity to a nucleotide that a static drawing might suggest

in three dimensions, the sugar is puckered and can exist in a 3’ end or c2 end conformation. If the 2’ is up= end, if down it is eco.

explain what is different for RNA nucleotides vs DNA nucleotides (hint c2 or c3 endo)

RNA= the 2’ position of the sugar has an OH so the c2 ends conformation creates steric problems with the nuclease so the c3 ends conformer preferred.

dna= the 2’ position is a hydrogen so steric problems not present. both confomers possible, but the c2’ ends conformation predominates.

explain the differences between c2 and c3 endo conformations

c2’= places the phosphates father part from one another, and this is the one associated with the B form dsDNA.

c3’= adopted in dsDNA, which results in a more tightly spaced backbone than in B form DNA and the double helix forma the A form structure that is more tightly wound.

explain why B form DNA reigns supreme compared to A form

B form conformation allows proteins access through its wide major groove, but A form is too tight for that. However because RNA prefers the 3’ endo RNA/DNA hybrids and dsRNA use A form structures.

nucleic acids can use non canonical ( not A:T or C:G pairing) to form alternative structures. explain those.

there are sun and anti conformations. syn has the base rotated to same side, and only Turin nucleotides can adopt this one. anti has then stacked like staircase, it is more energetically favorable because less steric repulsion.

are WCF base pairs anti or sin?

they are anti/anti with both pointed away form the base so they can interact with another and form h bonds.

what are Hoogsteen base pairs

syn/anti pairs that can be A:T, C:G, or G:G. far less common and allows 3-4 strands to be present in a helix. they are found in damaged DNA and DNA bound by drugs but new evidence suggests may form normally to regulate gene expression.

RNAs are normally ss in cells but can fold into complex structures, explain how

starting form the linear chain, the molecule forms WDF (or hoogsteen) base pairs with itself and the secondary structure packs together into a 3d fold with a core just like a protein.

how do nucleotide building blocks for nucleic acid polymers, like what is the one big step.

the 3’ OH attacks 5’ triphosphate

why is the genome- the principal storage- DNA and not RNA

RNA is too unstable with the 2’ hydroxyl group. RNA is more susceptible to degradation than DNA and it breaks down easily and rapidly. Another reason: the B form of DNA allows easier access information in DNA in major groove without need to unwind helix.

DNA consists of two single strands annealed together, what sets the stability of the two strand being annealed together versus separated as free strands.

The annealed form is great for long term storage but also need to separate the strands to be able to make use of base pairing for replication and transcription.

how do the annealed stands of DNA separate during replication

DNA is denatured during genome replication and then it melts at 5’ ends of a gene before it is expressed. helices unwinds

Compare the strength of A:T bonds to G:C bonds

A:T are weaker because they only form two hydrogen bonds whereas G:C form 3

Tmelting is what

the temperature at which the helix is half double stranded half single stranded so 50% denatured. Melting temp is related to dsDNA stability. Stable helix= high Tm, unstable helix= low Tm

what sets the melting temperature of dsDNA

the relative A/T and G/C content, more G/C makes it harder to separate the strands so needs higher temp.

what does the melting temp tell us in the lab

the temp that we need to go above to cause dsDNA to denature and the temp we need to dip below to get two strands to anneal together

how can the cell pack all this DNA inside itself, and what constraints does that packing impose on unwinding and accessing the genetic information stored inside?

it has to be compacted, cells exploit the topological properties of DNA to induce genome compaction

wound up materials have topological properties that constrain how they can be deformed, explain what those are

topology is the number of cross overs in the material, it means you can tug and twist on the knots all you want but you can’t change one into the other unless you cute the rope. The topology of DNA is described by linking number. TW is the twist and WR is the write

compare twist vs writhe

twist is around the axis, write is the central axis twists about itself.

If you have a Lk of 100, and a Tw of 50 and a Wr of 52, do you need topological properties?

yes because the number does not equal 100.

DNA in the favorable B form has an intrinsic twist to it, what does that mean

it is natural, so with one turn of the helix every 10.4 bp, the natural twist, aka Lk0, is given by the number of base pairs/ 10.4.

How do cells actually change the topology of their DNA?

they use topoisomerase enzymes to change DNA topology because the linking number itself cannot be changed by deformation of the structure, but if you were to cut the structure, add or remove some twist and reseal it you could change the linking number.

what are these steps of topoisomerase use to change DNA topology

1. cut a strand

2. manipulate the DNA strands around the cut to adjust the twist

3. now reseal the. two DNA strands

if you unwind a few turns, what happens to the DNA

the linking number decreases and it gets negatively supercoiled and less compact.

what is another case that topoisomerase are needed

to resolve supercoiling that develops when the genome is locally unwound for DNA replication or transcription.

how many orders of magnitude more material is there to cram in there for eukaryotic cells.

3 times more for eukaryotic.

what are the 5 types of histones that wrap around histone proteins to form nucleosomes

H1, H2A, H2B, H3, H4

after DNA wraps around histone proteins to form nucleosomes, what happens

they are further packed into a 30nm fiber which involves supercoiling

how does histone modification impact chromatin compaction

heterochromatin proteins such as HP1 bind across methylated histones to promote chromatin compaction

Euchromatin

Accessible region, transcription of genes can occur here and it is marked by histone acetylation

heterochromatin

inaccessible region, transcription of genes cannot occur here. marked by histone methylation and binding of heterochromatin proteins,

what is a nucleosome

DNA wrapped around histone proteins

what is replication

using a template strand to add complementary nucleotides

genome replication is semi-conservative, what does that mean

each daughter helix gets one strand form the parents and one new strand, the parent strand serves as a template to generate the daughter strand.

what is DNA polymerase/ explain it thoroughly

the enzyme that catalyzes nucleotide addition. it uses DNTPs as substrates, adds dNTPs that base pair with nucleotides in the original template DNA strand. It only adds dNTPs to the 3’ end ion the last nucleotide in the polynucleotide strand. 3’OH is essential for the reaction to proceed. synthesis can only proceed off an already existing double stranded fragment thus polymerase requires a short nucleic acid primer annealed to template strand to initiate synthesis

what is the structure of DNA polymerase

double helical geometry leading right up to the final 3’ OH so that the next nucleotide to be added via nucleophilic attack of the 3’OH on the dNTP is complementary to the template strand.

Mg2+ coordinated by DNA polymerase facilitates chemistry by …

activating the 3’ OH attack on dNTP phosphate which makes dNTP more electrophilic and stabilizes phosphates negative charge. if polymerase is denied metal it will not be able to preform chemistry

why does DNA polymerase require a polymer

base pairing interactions are more specific in the context of an existing double stranded structure than on their own

what is the major mistake DNA polymerase can make

occasionally a nucelobase will tautomerize into a rare form in which the position of the H bond donor acceptor are different. the donor and acceptor switch and changes base pairing

what do base pairing arrangements between rare tautomeric forms lead to

disincorporation into the growing DNAS strand.

DNA polymerase makes other mistakes caused by misfiring of a different nature

different but nontautomeric chemical forms of bases, or normal bases that nonetheless bond inappropriately. They are known as wobbles.

DNA poly has a 3’→5’ proofreading activity to correct mistakes, it has 2 activities what are they

1. DNA polymerase adds nucleotides via its polymerase domain and

2. removes mismatched nucleotides via its exonuclease domain

how is an incorrect base detected and corrected

a newly added base that is wrong will destabilize the dsDNA structure. so the end of the strand is more flexible allowing it to adapt a conformation that moves the 3’ end into the exonuclease site of the polymerase. once the 3’ end enters, the terminal nucleotide is removed. it has a second chance now.

what are all of the steps if replicating a genome

the dsDNA needs to unwind to copy both strands which requires energy as the double helix is a stable structure. polymerase also needs primer to extend off of so we need enzyme that can add primers, but if going to keep unwinding then in order to polymerize a region of the bottom strand we need another primer but then there are multiple on one strand that aren’t continuous.

DNA POLY 1 vs POLY3

1= not built for processivity built for accuracy over speed with high fidelity

3= built for sprees and built for proclivity

what happens at the Ori

DNA is melted and unwound by helices to produce replication bubbles that provide the ssDNA templates needed for synthesis

what does primate do to provide a primer to initate synthesis

synthesizes a short complementary RNA sequence and the DNA/RNA hybrid will be A-form allowing it to be detected and eliminated later.

within the replication bubble, two forks progress away in opposite directions, explain what happens

because DNA is 5’→3’, there is one leading strand and one lagging strand that is discontinuous and makes fragments

successful synthesis of the lagging strand requires several enzymatic activities, explain

addition of primer by primate. extension by DNA poly 3 until it encounters 5’ end of another Okazaki fragment and falls off. Primase and pol 3 continue to work tirelessly to make fragments ahead of fork. DNA pol 1 recognizes the RNA primers and removes them and replaces them with DNA sequence. Nicked DNA fragments sealed by ligase

how does DNA ligase seal nicks?

first dan ligase uses an ATP to adenylate itself on a lysine residue forming ligase-AMP. Now the AMP is transferred from DNA ligase to the 5’ phosphate of the nicked DNA strand. Now the 5’ end of the nick is charged with a good Leaving group (AMP) base catalyzed nucleophilic attack by 3’OH can seal the stand.

what do topoisomeraases do

deal with supercoiling that arises from unwinding

what do single stranded binding proteins do

keep ssDNA from reannealing

what happens when the replication fork gets to the ends of linear chromosomes

removal of the RNA primers leads to overhands that cannot be filled in and if they aren’t the chromosomes will get shorter and shorter with each round of replication. The enzyme telomerase will provide solution to shrinking ends

what are telomerases'/ what do they do

they are regions of repetitive sequences at the end of eukaryotic chromosomes. telomerase is a polymerase that carries an rNA fragment with it. It uses the RNA fragment to template DNA synthesis at the end of telomeres on the parental strand of DNA. once extended, the standard replication machinery can extend the DNA of the lagging strand which prevents chromosomes from shortening .