nucleic acids

Question 1

nobel prize winners

Answer 1

watson ,crick, and wilkins for nucleic acids

fire, mello for rna gene splicing

Question 2

information storage

Answer 2

stored and transmitted from one generation to the next via DNA

genes are the hereditary units of the chromosomes, which are long strands of DNA.

stored in the base-pair sequences of the DNA strands.

Question 3

nucleosides

Answer 3

Each nucleoside is an aldopentose sugar linked to a purine or pyrimidine base.

Question 4

dna structure

Answer 4

chain of 2-deoxy-D-ribose rings linked by 3’ to 5’ phosphodiester linkages

Question 5

rna structure

Answer 5

a chain of D-ribose rings linked by 3’ to 5’ phosphodiester

additional -OH group at the 2’ position of each ribose ring

Question 6

pyrimidines

Answer 6

six membered rings with two nitrogens and two carbonyl oxygens

cytosine and thymine

uracil similar to thymine but missing a methyl group

Question 7

purines

Answer 7

five membered ring fused to a six membered ring

adenine and guanine

Question 8

glycosidic linkaes

Answer 8

C-N bond replaces the N-H bond in the free nucleotide bases

ribose sugars by a C-N bond at the C-1’ position of the ribose ring, in place of the -OH group.

Question 9

5’ and 3’ nucleotides

Answer 9

phosphate is attached to the sugar at the 3’ position then it is a nucleotide-3’-phosphate

Question 10

oligonucleotides

Answer 10

link connects the 3’ position on one nucleotide to the 5’ position on another nucleotide through a phosphodiester linkage

Question 11

heterocycles

Answer 11

nucleotide bases have nitrogen atoms as part of their rings, so are heterocyclic

Question 12

bronsted lowry bases

Answer 12

nitrogen atoms can gain a proton and become charged

Question 13

acid base equilibria

Answer 13

nitrogen atoms tend to form three bonds, leaving a ‘lone pair’ of electrons that can act as a brønsted-lowry base

Question 14

pka and base strength

Answer 14

large pKa value indicates that an equilibrium lies to the left hand side.

indicates strong base

Question 15

pyrrole

Answer 15

weak base, nitrogen lone pair is involved in pi bonding

nitrogen is sp2 trigonal planar with one p orbital containing two electrons - aromatic - more stable

nitrogen lone pair is not available for bonding

Question 16

nucleophiles

Answer 16

pyridine and imidazole have nitrogen lone pairs, so they are nucleophilic and can react with electrophiles, such as methyl iodide

chemical carcinogens are electrophilic and react with the nitrogen lone pairs of the basic nitrogens of the nucleotide bases.

Question 17

tautomerisation

Answer 17

transfer of a hydrogen atom from one place to another, within the same molecule, resulting in a different structure.

Question 18

lactim and lactam

Answer 18

lactam is present in vast excess

Question 19

amino and imino

Answer 19

amino is present in vast excess

Question 20

hydrogen bonds

Answer 20

functional groups with lone pairs of electrons are classified as hydrogen bond acceptors

functional groups with an O-H or N-H bond are classified as hydrogen bond donors.

if hydrogen bond donor and acceptor groups line-up correctly on pairs of molecules then the hydrogen bonding is said to be complementary.

Question 21

tautomerisation in nucleic acids

Answer 21

donors - acceptors

acceptors - donors

leads to incorrect base pairing, and changes DNA sequence which can be carcinogenic

occurs once every 10^4 base pairs copied

human genome is ∼ 3 × 10^9 `base pairs in length, this would lead to ∼ 300 000 errors every time the cell divided

actual in vivo error rate is one in 10^8 base pairs copied, due to the presence of proof reading enzymes

Question 22

alkoxy groups

Answer 22

OR

Question 23

phosphodiester linkages

Answer 23

in phosphate esters it is possible to replace all three hydroxy groups (OH) on the phosphorus with alkoxy groups (OR) of an alcohol

Question 24

phosphodiester hydrolysis

Answer 24

phosphate esters are relatively stable and their hydrolysis requires harsh conditions

Question 25

phosphodiester hydrolysis in rna

Answer 25

rna is much less stable to hydrolysis than DNA, due to the presence of an -OH group at the 2’ position of the ribose ring

Question 26

sugar base link cleavage

Answer 26

sugar-base link (a C-N bond) is unstable to acid hydrolysis, especially in the purines. Protonation at N-7 aids the hydrolysis with the loss of the nucleotide base to give a basic DNA

Question 27

deprotonation

Answer 27

phosphate groups are deprotonated at physiological pH, meaning that polynucleotides are negatively charged (polyanions)

Question 28

earlt theories of DNA

Answer 28

Chargaff’s rules - by hydrolysing DNA, separating and analysing the resulting bases it was determined that DNA has an equal number of adenine and thymine bases (A = T) and an equal number of guanine and cytosine bases (G = C). NMR and X-ray data - correct tautomeric forms of the bases were determined. X-ray diffraction photographs - information that DNA is helical.

Question 29

B-DNA

Answer 29

most common form conditions for high stability B-DNA are high humidity(>92%), as found in aqueous environment presence of metal cations (Na+) which interact with the negatively charged phosphate groups

Question 30

watson-crick structure

Answer 30

two deep grooves between the sugar-phosphate chains, a relatively narrow minor groove and a relatively wide major groove, unequal sizes because deoxyribose rings are assymetric, and the top edge of each base pair is structurally different from the bottom edge aromatic bases occupying the central core of the helix, forming complementary base pairs (GC, AT), essentially planar - stack on top of one another, planes are nearly perpendicyular to the helical axis (6% tilt) a right handed, antiparallel, double helix formed by two sugar-phosphate chains wound about a central helical axis

Question 31

B-DNA dimensions

Answer 31

helix diamter is 20 angstroms base pair has a thickness of 3.4 angstroms helical pitch is ~34 angstroms human genome is 1.02m

Question 32

A-DNA

Answer 32

A-DNA is also a right handed, antiparallel double helix. A-DNA is a wider and flatter double helix than B-DNA, with a helix diameter of ~ 26 Å. The helical pitch is similar to B-DNA at ~ 34 Å, but there are 11.6 base-pairs per full turn of the double helix. This means that the aromatic bases have a closer contact, which favours crystal formation.

Question 33

A-DNA structure

Answer 33

The planes of the base-pairs in A-DNA are tilted by ~20o with respect to the helical axis. The base-pairs are displaced away from the helical axis, leading to a central helical cavity. The major groove of A-DNA is very deep and the minor groove is very shallow.

Question 34

Z-DNA

Answer 34

forms when the concentration of cations (typically Mg 2+) is high interact with the charged oxygen atoms which are closer than in B-DNA (8 angstroms vs 12 angstroms) left handed, antiparallel, double helix sugar phsophate chains zig zag down the outside of the double helix

Question 35

Z-DNA dimensions

Answer 35

helix diamter is ~18 angstroms helical pitch of ~44 angstroms 12 base pairs per full turn found in local segemnts of B-DNA where the DNA is interacting with proteins (enzymes that repair and modify the bases)

Question 36

denaturation

Answer 36

leads to changes in the DNA’s physical properties (eg. viscosity decreases) and spectroscopic properties (UV-vis absorbance increases) absorbs strongly in the wavelength range 200 - 300 nm, but each nucleotide base has its own absorption profile aromatic systems of the purine and pyrimidine bases undergo electronic transitions (involving their p electrons) when a photon of the correct wavelength, l, is absorbed

Question 37

hyperchromic shift

Answer 37

base pairs become unstacked free nucleotide bases have a ~40% higher absorbance rate

Question 38

relative absorbance

Answer 38

denatured - 82 degrees, absorbance at 250nm is ~0.4 native - 25 degrees, absorbance at 250nm is ~0.3 mid-point of the melting curve is known as the melting temperature (Tm).

Question 39

stabilty of DNA towards denaturation

Answer 39

the solvent ions present pH DNA composition (percentage GC vs AT) GC base-pairs have three hydrogen bonds, compared to just two in AT base-pairs. Thus, more energy is required to separate GC base-pairs.

Question 40

quenching

Answer 40

denatured DNA is rapidly cooled resulting DNA will only be partially paired, as probability of pairing in the correct order is almost zero

Question 41

renaturation

Answer 41

denatured DNA is heated to a temperature ~25 oC below its Tm and held at that temperature for long enough, the DNA will eventually ‘renature’ process involves continual melting and re-forming of short base-paired regions (annealing). enough thermal energy is present to separate weakly bound (incorrect) base-pairs, but there is not enough thermal energy to completely denature the DNA

Question 42

local breathing

Answer 42

at moderate temperatures, short sections of DNA can separate (they no longer associated by hydrogen bonding) for a short period of time, before re-associating

Question 43

mutations

Answer 43

during DNA local breathing, at the separation site, the base pairs are not hydrogen bonded. The hydrogen bond acceptor groups of the nucleotide bases can now behave as nucleophiles and may react with electrophilic chemicals present can cause mutation of the DNA and disrupting the base pairing and can cause the incorrect base to be incorporated during replication

Question 44

circular DNA

Answer 44

double helical DNA can form a closed loop by covalently bonding the 5’ and 3’ ends of both strands (to each other)

Question 45

superhelical DNA

Answer 45

circular DNA can be twisted further, leading to highly strained states (akin to a wound up rubber band). These forms of DNA are said to be supercoiled, supertwisted, or superhelical. two forms of superhelical DNA that can only be interconverted by breaking and re-forming covalent bonds are said to be topoisomers enzymes responsible for converting DNA from one topoisomer to another are called topoisomerases.

Question 46

supercoiled DNA in the cell nucleus

Answer 46

supercoiled DNA interacts favourably with proteins called histones. the complex formed is called chromatin. further coiling of the DNA spacers in chromatin produces a compact chromatin found in the cell nucleus strain energy present in supercoiled DNA is used to drive some biological processes

Question 47

topoisomerase inhibitors

Answer 47

molecules that interfere with the function of topoisomerases (topoisomerase inhibitors) are effective antibiotics and cancer chemotherapy agents

Question 48

intra strand base pairing

Answer 48

RNA can fold back on itself via intra-strand base pairing to create a variety of folds, bends (hairpin turns), U-turns, loops, bulges and junctions.

Question 49

rna helix formation

Answer 49

steric repulsion between the 2’-hydroxy and the C-3’ oxygen and C3’-phoshodiester means that RNA does not form the B-form but can form short lengths of the A-form

Question 50

parental and daughter strands

Answer 50

parental DNA double helix must be unwound to expose base pairs that are not hydrogen bonded. this requires another class of enzyme (type IIA topoisomerases - some of which are called DNA gyrase) point at which double stranded (parental) DNA is unwound is called a ‘replication fork'

Question 51

transcription

Answer 51

several turns of the DNA double helix unwind to form a ‘bubble’ that exposes the bases of the two strands

Question 52

nucleotide monomers

Answer 52

nucleotide monomers in rna synthesis are nucleotide triphosphates ATP adenosine triphosphate CTP cytidine triphosphate GTP guanosine triphosphate UTP uridine triphosphate

Question 53

rna chain growth

Answer 53

pyrophosphate[P2O7]4- is released as the next nucleotide base is added to the growing rna chain

Question 54

ribosome

Answer 54

two thirds rrna and one third protein evidence that the rRNA of the ribosome catalyses the peptide bond formation rather than the protein part ribosome binds mRNA, which serves as a template to pass on the genetic information mRNAs can be regarded as a series of consecutive 3-nucleotide sequences, known as codons. Each codon specifies a particular amino acid

Question 55

trna

Answer 55

tRNAs deliver amino acids to the ribosome. Each tRNA carries one amino acid which is covalently linked at the 3’-hydroxy position single stranded tRNA is folded into a clover leaf secondary structure with 3 loops and at least 4 regions with complementary base pairings on the middle loop, opposite the 5’ and 3’ ends there is an anticodon sequence of three nucleotides that is complementary to the codon sequence

Answer 56

ribosome binds mRNA and this facilitates the binding of multiple tRNA molecules, through complementary hydrogen bonding (between codons and anticodons ribosome catalyses the transfer of an amino acid from one tRNA molecule to another, which increases the length of the growing polypeptide by one amino acid once tRNA has passed its amino acid to the growing polypeptide, it is ejected

Question 57

rna self splicing

Answer 57

self-splicing involves the removal of a central segment (intron) from an rna strand and joining together the resulting end portions (exons) without an external catalyst a 421-nucleotide intron is removed from a 6.4 kbase precursor to give the mature RNA.

Question 58

human genome project

Answer 58

first draft was in 2000 complete sequence was published in 2003 allows us to manipulate and harness the genetic information

Question 59

dna sequencing

Answer 59

dna molecules are too large to be sequenced intact, so must be cleaved into fragments enzymes that cleave the dna at specific base pair sequences are called restriction endonucleases

Question 60

base sequence specific cleavage

Answer 60

each of the 200 known endonuclease will cleave DNA at well defined points in the chain

Question 61

solving the puzzle

Answer 61

cutting the dna with other enzymes with different selectivity will lead to partial overlap these overlaps must be identified to be able to determine and complete the dna sequence sanger method is used in sequencing machines

Question 62

sanger method

Answer 62

ecoli dna polymerase I is used to synthesize complementary copies of the single stranded dna fragments being sequenced only one fragment at a time each fragment is incubated with: dna polymerase I a dna primer (klenow fragment) four dntp monomers a small amount of dideoxynucleoside triphosphate(ddNTP)

Question 63

ddNTPs

Answer 63

missing an OH group at the 3’ position, meaning that no phosphate group can be attached can be radiolabelled or labella by attachment of a fluorescent dye

Question 64

automated sequencing

Answer 64

requires fluorescent labeling all of the chain growth reactions are carried out in a single vessel analysed by gel electrophoresis terminal base is identified according to its characteristic fluorescence(diff colours for each ddNTP) large numbers of machines are used >100

Question 65

uv and radiation damage

Answer 65

generate hydroxy radicals from water with dioxygen, peroxy radicals are formed large concentration of water and dioxygen in biological systems