BIOMG 3320 Prelim 1

Why is RNA less stable than DNA?

RNA can go under Alkali hydrolysis where the OH- in dilute base can attack the 2' OH of the ribose. The 2' OH acts as a nucleophile in an intramolecular displacement producing a 2', 3'- cyclic monophosphate derivative and a shortened RNA || Pretty much, the 2' OH group of ribose can easily attack the phosphate backbone and break into 2 pieces

What are similarities and differences between DNA and RNA?

Differences || DNA is usually double standard and therefore rigid, while RNA is single stranded and flexible || The nucleotides of DNA are AGTC, the nucleotides of RNA are AUGC || RNA has a 2' OH, therefore it is susceptible to alkaline hydrolysis, while DNA is not. The 2' OH is a reactive group that confers catalytic activity to ribozymes??

How do adjacent nucleotides join together?

The 3' hydroxyl group joins to the 5' end of the other

What are the functions of nucleotides and explain each one?

In general, they function in energy metabolism and as signal molecules || Nucleoside 5' triphosphates are carriers of energy || ATP is central to energy meta || GTP drives protein synthesis || Granny Pees || CTP drives lipid synthesis || Crystal Licks || UTP drives carbohydrate metabolism || Ugly Clav Mogged

What is the major contributor to the thermostability of dsDNA?

Base stacking interaction

What are the key discoveries in DNA structure

1.) Chargaff's pairing rule of nucleic acid where within one specifies, the amount of A's equals T's , and G's equal C's (A=T, G=C) || 2.) Rosalind Franklin and Maurice Wilkins DNA X-ray diffraction pattern || Where an X pattern indicates a helical repeat structure and two different periodicities || From diffraction patterns, you can extract geometric patterns//measurement about the DNA molecule

What is the distance between nucleotides

3.4 Angstroms

Is DNA positively or negatively charged?

Negatively charged due to the sugar phosphate backbone

What are the forces that stable the DNA double helix?

Base stacking interactions || The major contributor to the thermostability of dsDNA || Hydrogen bonding between complementary base pairs does not contribute to the thermostability of dsDNA || Metal cations shield the negative charges of backbone phosphates(which cause instability) and stabilize dsDNA

What does not contribute to the thermostability of dsDNA?

hydrogen bonding

What type of DNA is more stable (GC rich or AT rich)?

GC rich DNA is more stable because GC pairs have more base stacking interactions, not because they have more H-bonds

Describe the different structural forms of DNA

All of the forms have the same watson and crick base pairing (A=T, G=C) || The A form occurs in dehydrated conditions (DNA becomes more condensed, pitches shorten, and diameter increases)(right handed) || The B form is the most dominant form under physiological conditions (right handed) || The Z form is left handed and occurs in short stretches. It may also regulate gene expression

Describe the various unusual structures certain DNA sequences may adopt

Palindrome || Read the same backwards as it is read forwards || Can be recognized by DNA binding proteins || Can form hairpin structures by base pairing within the same DNA strand || Mirror repeat || Mirror symmetry around a certain point || Hairpins || Palindromes can form these by base pairing within the same DNA strand || Cruciforms || Double stranded palindromes can form these || One hairpin on either strand, the hairpins oppose each other to form a cross shape

What is Hoogsteen pairing?

DNA structures formed by three stands || Non-watson-crick pairing || It is denoted by * || The triple helical DNA contains two pyrimidine strands and one purine strand

Where are triple helices found?

In regions involved in gene regulation, but overall pretty rare || Also called H-DNA

What type of base pairing is found in triplex DNA?

Hoogsteen pairing

Describe RNA base pairing

RNA is single stranded but lots of short stretches of complementary sequences found within the same molecule will base pair to form the secondary (A form) and tertiary structures || Hydrogen bonds, watson and crick pairing,and hoogsteen pairing can readily form

What are the differences in RNA base pairing as compared to DNA base pairing?

RNA has Uracil while DNA has Thymine || G=U base pairing is allowed in RNA (still rare)

Describe the complex 3-D structures of RNA

The predominant double stranded structure is an A form right handed double helix || Bulge,, internal loops, and hairpin loops are often seen in RNA structures || G-U base pairing is allowed in RNA || tRNAs are an example of RNA 3D structure || Have many unusual H -bonding patterns not seen in DNA || Unusual base-pairing patterns are frequently seen in RNA || Hydrogen bonds can be formed between the base and phosphate ribose backbone

Describe the three states of DNA during Denaturation???

Under heat or high pH, the rigid rod of double helical DNA denatures into partially denatured DNA. || Then under more heat or high ph, separation of strands occurs where there is now no regular backbone and the bases are not stacked || The dan is separated into stronds in random coils

What allows DNA to be able to be separated into separate stranded?

A lack of covalent bonds || Under heat or high pH , the ds structure can be disassembled into two single strands

How does denaturation happen?

Gradually, with partially denatured DNA occurring as an intermediate

What is renaturation?

When the temperature is lowered, dsDNA can be reestablished by complementary base pairing

Describe dna in terms of viscous or sticky and explain why we don't use this method?

Double stranded purified DNA in solution is viscous or "sticky || Denatured single stranded dNA is less viscous || We don't use this method because it is not a quantifiable measure of the extent of DNA denaturation

What is the quantitative approach for measuring DNA denatured states?

Using UV absorbance/absorption || All nucleotides have strong absorption around 260 nm, but different nucleotides have different absorption profiles || Denatured DNA has a 40% increase in UV absorption at 260 nm relative to double stranded DNA

How do we measure the melting temperature of DNA?

Through UV absorption. UV absorption is measured many times while gradually raising the temperature ( from 70 degrees C to 100 degrees C) until all DNA is single stranded, which generates a "denaturation curve"

Describe the two different states of DNA

The hypochromic state denotes decreased UV260 nm absorption in stacked bases (double stranded dna) || The hyperchromic state denotes increased UV260 nm absorption by 30-40% in unordered bases of denatured DNA

What is the melting temperature

It is the sharp transition point along the denaturation curve in which half of the DNA is president as separated single strands

What is the melting temperature dependent on?

1.) the G + C content of the DNA molecule and size || DNAs with more GC contents are more stable,and thus will have higher melting temperatures || ***This is due to stronger base stacking interaction contributed by GC pairs, NOT hydrogen bonding) || The melting temperature can be used to predict the base composition of DNA || (More GC bonds means higher melting point) || 2.)The pH and ionic strength of solution also affect the stability of DNA || Cations stabilize the helix through electrostatic interactions with phosphate backbones || A higher cation content (ionic strength) can increase the melting temperature of DNA

How are land markers on DNA generated/ how can we probe local DNA composition?

Through partial denaturation || Regions that are more prone to denaturation form bubbles of single stranded DNA within a double stranded DNA molecules during partial denaturation || The bubble regions correspond to more AT rich regions (LOWER GC content)

What are the nucleotide nonenzymatic transformations

Alkali hydrolysis of RNA || Deamination || depurination/depyrmidiation || Cross -linking

What is alkali hydrolysis of RNA

Alkali hydrolysis of RNA occurs when the 2' OH undergoes an intramolecular reaction with the phosphate backbone, creating two separate pieces forming a cyclic phosphate structure and a shortened RNA? || RNA has a 2' OH, therefore it is susceptible to alkaline hydrolysis, while DNA is not. The 2' OH is a reactive group that confers catalytic activity to ribozymes??

What is Deamination?

Cytosine amine group can get attacked by water molecules, which converts the amine group (pos 4) to a carbonyl group || As such, cytosine is converted to uracil upon deamination (which is why we do not have uracil in our DNA) || In addition, cytosine can get methylated to form 5-methylcytosine. Deamination of 5-methylcytosine leads to the formation of thymine

Why are DNA repair mechanisms so crucial?>

Because cytosine deamination occurs to about 100-500 cytosines per cell per day || ...

Why don't we have uracil in our DNA???

Because cytosine can spontaneously deaminate into uracil || If uracil was used in DNA, this would cause massive mutations, but because it is not, repair machinery can easily identify and fix uracil as an error

What is Depurination?

The N-beta- glycosidic bond can get broken by water (hydrolyzed). This leads to the formation of an AP site (apurinic/apyrimidic) within the DNA backbone and a free purine/pyrimidine || There are DNA repair mechanisms that can reinsert the correct base into an AP site || prelim2???

? Why does DNA contain Thymine rather than Uracil?

The slow cytosine deamination || In DNA, the product of cytosine deamination , uracil, is readily recognized as foreign DNA, and is removed by a repair system || If DNA normally contained Uracil, recognition of uracils resulting from cytosine deamination would be more difficult, and unrepaired uracils would lead to permanent sequence changes

What is Cross-Linking?

Adjacent thymines can be crosslinked by UV to form a cyclobutene thymine dimer or a 6-4 photoproduct || Cross-linking occurs randomly || There is no way to predict whether a thymine dimer or 6-4 photoproduct will be produced

What is a nucleotide enzymatic transformation?

Methylation

What is methylation?

Methylation occurs to some bases of DNA || Methylated cytosine, 5-methylcytosine, and methylated adenosine, N6-Methyladenosine, are generated by specific enzymes at specific sites for the purpose of signaling || Methylation usually occurs within a certain sequence or region of DNA || Methylation can be a defensive mechanism , where enzymes can differentiate between host and foreign DNA based on methylation patterns, or play a role in DNA repair and gene expression!

How long is the human genome in meters

1.02 meters || The nucleus is only a few micrometers

Describe the major topological problem of DNA and how it is generated?

DNA has to be replicated, which requires the separation of double stranded DNA into single strands || Further continuous strand separation requires rotation of the rest of the DNA helix which can create more tension in the wound DNA (supercoiling) || Tension in the DNA occurs at the replication fork, and the cell needs a way to relieve that tension

Does DNA topology use an open DNA molecule?

no , it always involves either a circular DNA or a long dsDNA with fixed ends

What is DNA supercoiling?

Supercoiling is the coiling of a coil. It is a way to relax topological stress/tension || DNA supercoiling is important for DNA compaction into the nucleus || DNA supercoiling does not break any chemical bonds || Topological stress causes DNA supercoiling, which is a way to relax the topological stress

What is Linking Number (Lk)

Linking number is a topological property that measures supercoiling || Lk= number of times one strand winds around the other in dsDNA || It is a topological number that has to be an integer number

What is Lk0

Lk0 is the linking number of B form DNA in its relaxed state || Lk0= number of base pairs (bp) divided by 10.5 bp || Example || If a circular DNA of 2100 bp is relaxed, its Lk0 is 200

How many times will DNA wind around itself in a relaxed state?

It will wind around itself every 10.5 bp so for every 10.5 bp, Lk increases by one!

How does supercoiling of DNA tend to form?

When the two strands of a circular DNA duplex are wound around each other more or less than once every 10.5 bp(unstable)

What state is cellular DNA in?

Cellular DNA is underwound and negatively supercoiled

What produces negative supercoils?

DNA underwinding produces negative supercoils

What produces positive supercoils??

DNA overwinding produces positive supercoils

What is Twist (Tw or T)

T= twisting number || It is the number of turns resulting from base pairing in B form DNA || Or Number of bp divided by 10.5 bp in B form DNA || Twist is a geometric number and can be any real number. || It shows you how the base pairs are stacked depending on state

What is Tw if all base pairs point in the same direction,

Tw= 0

What is Tw if you twist the DNA once

Tw= ½ || This is because ½ of the base pairs point in one direction, while the other ½ of the base pairs point in the other direction OR || Because a twist is a half turn

What is Tw if you twist the DNA twice?

Tw=1 because it generates one complete turn

What happens when Tw does not equal to 0

The neighboring base pairs lay within different planes, which affects the stacking interactions within DNA

What is Writhe? (W)

W= writhing number || It is number of superhelical turns || (it is a geometric number and can be any real number

Can Twist and Writhe change?

Yes, if the shape of the object changes, even though the topology of the object has not changed????(what does this even mean)

What happens if you add Twist and Writhe together?

It will be an integer number, which is the Lk (linking number0 || Which is why Tw and W don't have to be integer numbers...

What determines the degree of supercoiling?

Linking number || By changing the linking number, we can change the supercoiling state of DNA || The base pairing interaction between the two strands of a DNA molecule creates force to maintain the Tw number the same as in its relaxed state || Lk=Tw + W

What state does DNA want to always be in/go back to?

To where every base pair contributes equally to twist (1/10.5 bp)

What does the positive/negativge sign of Writhe tell you about the topology of DNA?

A negative writhe means that Lk

What is superhelical density?

It measures DNA supercoil independent of the length of the molecule || It corresponds to the number of turns removed or added relative to the number present in relaxed state || Sigma = change in Lk/ change in Lk0 OR || Sigma = (Lk-Lk0 ) / (Lk0)

What is the superhelical density in cells?

5-7% or -0.05 to -0.07

What are Topoisomerases?

Topoisomerases catalyze changes in DNA by changing the linking number, which corresponds to the supercoiling state of DNA || They are enzymes that regulate the topological state by changing the linking number

What are topoisomers?

They are two forms of circular DNA that differ only in a topological property such as linking number || I.e each band in a gel electrophoresis of supercoiled DNA treated with topoisomerase

When separated by gel electrophoresis, does supercoiled DNA or relaxed DNA run faster?

Supercoiled DNA runs faster than relaxed DNA

What happens if you treat supercoiled DNA with topoisomerase

It will relax

How does Type 1 topoisomerase work? (bacterial type 1 topoisomerase)

It breaks one strand and increases linking number (LK) by steps of 1 || 1,) It will cleave one strand through a Tyrosine Hydroxyl group that attacks the phosphate phosphodiester bond in the backbone of DNA || No ATP is required || 2.) It will pass the unbroken strand through the break || 3.) It will ligate and close the break by releasing the Tyrosine Hydroxyl group and resealing the phosphodiester bond/DNA backbone || This results in a change of linking number by n+1, || where n=the original linking number before the break || *In general, every time you pass a strand through a break, you increase the linking number by 1

How does Type 2 Topoisomerase work

It breaks both strands and changes the linking number by 2 || It cleaves both strands of DNA, requiring ATP || Changes linking number in steps of 2

What is the difference between Type 1 and Type 2 topoisomerases?

Type 1 topoisomerases cleave one strand of DNA,, changes the Lk by 1, and does not require ATP while Type 2 topoisomerases cleave both strands of DNA, requires ATP, and changes the Lk by 2

Why are Topoisomerases important?

They are essential in DNA replication, transcription, and packing || They function in DNA replication to separate DNA strands. Separation at the replication site causes topological stress ahead of the replication site || Topoisomerases are also drug targets

Why are Topoisomerases drug targets?

Because without topoisomerase, cells cannot replicate DNA or transcribe genes || Used in antibiotics to kill bacteria || Also used in cancer therapy to kill highly proliferative cells. Since these cells replicate so fast and are undergoing DNA replication. They are more susceptible to topo inhibitors

What form is nuclear DNA in in nondividing cells

Chromatin

What is chromatin?

Chromatin is a complex of DNA and proteins that forms chromosomes within the nucleus of cells

What is the DNA packing problem?

Human DNA stretches 2m but it must be packed in a nucleus which is only 5-10 micrometers

In an electron microscopy image of a nucleus what are the dark vs light areas?

The dark areas are condensed chromatin called heterochromatin which are associated with silent genes/nontranscribed genes. || The light areas are less compact chromatin called Euchromatin and associated with transcriptionally active genes

What are nucleosomes?

Nucleosomes are the basic structural unit of eukaryotic chromatin. It consists of a "bead" and the adjacent linker DNA

What are beads on a string model

It describes nucleosomes where DNA will wrap around proteins which aid in DNA compaction

How much DNA is contained in one nucleosome?

About 200 bp. This is the linker DNA with the DNA that wraps around the nucleosome

What are the 2 main components of nucleosomes?

Histones, which are proteins that form the nucleosome core which DNA wraps around, and Linker DNA between nucleosomes

What are Nucleases

Enzymes that degrade DNA or RNA

What are DNases

Nucleases that are specific to DNA and not RNA

What are Exonucleases

They are nucleases that degrade nucleic acids from one end of the molecule (either 5' to 3' or 3' to 5')

What are Endonucleases?

Nucleases that degrade nucleic acids at the specific internal sites to generate smaller fragments

What is the light digestion experiment and what is it used for?

It is used to determine how much DNA is around the nucleosomes. || First, they cleave chromatin (beads on a string) with light (gentle) digestion. This will cleave apart the histones and some linkers. Some linkers are protected and some are accessible, leading to variety of sized fragments || Second, they separate the fragments by gel electrophoresis || Some fragments have the DNA wrapped around one nucleosome while other fragments have multiple nucleosomes and contain the adjoining DNA linker || Third, after light digestion, we find that the smallest amount of DNA wrapped around nucleosomes is 200bp

What is the extensive digestion experiment?

First, only the DNA wrapped around the nucleosome is protected, so all the linkers are cleaved off. || Second, this will give one fragment that you can visualize by gel electrophoresis which we will see only one 140 nm band || Thus, we find that about 140 nucleotides are in contact with the nucleosomes proteins and about 60 are in the linker between nucleosomes

How did they find out what proteins make up the nucleosome core?

They dissociated DNA from the histone core using a high salt solution. THey found that there are FOUR types of histones in the core (H2A, H2B, H3, H4) AND there are TWO of each, forming an octameric structure.

What is something unique to histones about their makeup?

30% of the amino acids are the very basic lysine and arginine, making these proteins very positively charged?

Why is the makeup of histones with lysine and arginine important?

Because DNA is negatively charged, which creates favorable electrostatic interactions between the DNA and histones

What type of sequence conservation (similarity) do histones have with different eukaryotic species

High, they all wrap their DNA around histones

What is the atomic structure of nucleosomes?

Amino acid tails of the histones stick out. DNA wraps around the histone coil about 1.65 times. DNA binding to the histone core is facilitated by hydrogen bonding

What type of supercoiling does the assembly of nucleosomes create?

Negative supercoiling. Therefore, there must be a change in linking number. Topoisomerase is used to change the linking number and "relax" the DNA

How are eukaryote topoisomerases different from bacterial gyrases?

Eukaryote topoisomerases cannot introduce negative supercoils, but they can relax positive or negative supercoils, depending on the enzyme.

How do eukaryotes get underwound DNA when they lack enzymes that underwind DNA?

Because of the protein spool (the histone core) used for packing of DNA into chromatin in eukaryotes, negative supercoils are introduced by the relaxation of positive supercoils

How many times do nucleosomes compact DNA and what is the problem with this?

Nucleosomes compact DNA only 7x but DNA must compact 10,000x to fit in the cell

What is the solution to nucleosome packing?

Nucleosomes pack into higher order structures after the addition of H1, which forms a 30 nm fiber. || For the rest of the compacting, nucleosomes continue to be packed further and form higher order structures, though it is not well understood. Bt, it seems to involve loiling and loops of rosettes || requiring the help of other proteins such as structural maintenance of Chromosomes proteins (SMCs)

What types of modification do histones undergo that affect the properties of chromatin

Covalent modifications such as methylation, acetylation, phosphorylation, glycosylation, etc.

What do these covalent modifications affect

They may affect the net charge, shape and other properties of histones, and they play a role in the regulation of transcription

What are histone variants and why are they important?

There are histone variants that can be swapped into the histone core by histone exchange factors. They are important because they can preferentially bind to DNA at different sites.