2. Nucleic Acids Flashcards
what are the types of nucleic acids, what are they composed of
Types of nucleic acids: DNA, RNA
composed of nucleotides:
- contains a base - Adenine, cytosine, guanine, thymine, uracil
- a sugar - deoxyribose and ribose
- a phosphate
the structure of nucleotides incorporated into the polymer determines the structure of the nucleic acid
DNA
A polymer of deoxynucleotides whose sequence of bases encodes genetic information in all living cells
RNA
A polymer of ribonucleotides, such as messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA)
nucleotide
- compound consisting of a nucleoside esterified to one or more phosphate groups
- monomeric units of nucleic acids
nucleoside
compound consisting of a nitrogenous base linked to a 5-carbon sugar (ribose or deoxyribose)
deoxynucleotide
nucleotide in which the pentose is 2’-deoxyribose
2 common sugars
deoxyribose and ribose
bp and kb
bp - base pairs
kb - kilo base pairs (1000 base pairs)
oligonucleotide
A polynucleotide consisting of a few nucleotide residues
B-DNA
The standard conformation of double-helical DNA
stacking interactions
The stabilizing van der Waals interactions between successive (stacked) bases in a polynucleotide
melting temperature (Tm)
The midpoint temperature of the melting curve for the thermal denaturation of a macromolecule.
For a lipid, the temperature of transition from an ordered crystalline state to a more fluid state
denaturation and renaturation
denaturation: the loss of ordered structure in a polymer
i.e disruption of native conformation (unfolded polypeptide), unstacking of bases, separation of strands in a nucleic acid
renaturation: the refolding of a denatured macromolecule so as to regain its native conformation
anneal
to allow base pairing between complementary single polynucleotide strands so that double-stranded segments form
Distinguish between properties of purine and pyrimidine bases.
Purines: Adenine (A), Guanine (G)
- double ring structure, basically planar, slight pucker in purine base
- poorly soluble in water
- largely hydrophobic with some polar groups (ability to form H-bonds)
Pyrimidines: Cytosine (C), Thymine (T), Uracil (U)
- heterocyclic
- aromatic, electron delocalization
What are the 5 common bases? Which are classified as pyrimidines and which are purines?
Purines: Adenine (A), Guanine (G)
Pyrimidines: Cytosine (C), Thymine (T), Uracil (U)
T - DNA
U - RNA
Is the sugar phosphate backbone polar or non polar?
polar
Name and distinguish between the two sugars that are incorporated into nucleotides
Ribose: found in ribose, has a -OH attatched to the 2’ carbon of the sugar ring in replace for a H present in deoxyribose
Deoxyribose: found in DNA, lacks -OH on the 2’ carbon (deoxy)
- both are furanose rings (5 membered with an oxygen)
naming of nucleosides and nucleotides
Nucleosides:
-
-osine in purines replaces -ines
(eg. adenine + ribose = adenosine) - pyrimidines → end in -idine
(eg. cytosine + ribose = cytidine) -
deoxy- is added as a prefix for nucleosides with deoxyribose sugar
(eg. thymine + deoxyribose = deoxythymidine
Nucleotides:
* nucleoside name + “5’ ____ - phosphate”
* ____ is the prefix for amount of phosphate’s in the phosphate group (mono-/di-/tri-/tetra-)
* phosphates attatched to multiple carbon/OH groups are described using bis-/tris- prefixes along with including the atom number that the phosphate is attatched to
(eg. adenosine 2’, 5’ bisphosphate)
5’ refers to carbon in the sugar ring, most of the time attatched to 5’ carbon but can be C2 or C3 as well
compare the structure of nucleosides with the structure of nucleotides
- nucleosides: sugar + base, no phosphodiester bond
- nucleotide: sugar + base + phosphate group, contains phosphodiester bond
Define the term nucleic acid
- also known as a polynucleotide
- polymer of nucleic acids
- major nucleic acids: deoxyribonucleic acid (DNA), ribonucleic acid (RNA)
explain why nucleic acids are said to have a “sense of direction”
- phosphodiester bonds in nucleic acids create asymmetric structures with a “sense of direction” →
- phosphodiester bonds link the 3’ carbon of one sugar and the 5’ carbon of the next sugar → creates a repeating sugar-phosphate backbone.
5’ end → 3’ end
What are the 5’ and 3’ ends of nucleic acid base sequences?
- The 5’ end of the nucleic acid chain has a free phosphate group attached to the 5’ carbon of the sugar.
- The 3’ end has a free hydroxyl (-OH) group attached to the 3’ carbon of the sugar
- nucleotides joined by phosphodiesters are described as residues
What is the primary (1°) structure for a nucleic acid?
the sequence of nucleotide residues; the order of monomers in a polymer
abbreviations for primary structures of nucleic acids
based on the first letter of each nucleotide written in sequence from 5’ → 3’ by convention
e.g: 5’ - ATGCAATG - 3’
can also be written as:
ATGCAATG, 5’-dAdTdGdCdCdCdTdG-3’, dAdTdGdCdCdCdTdG (DNA)
- T indicates the nucleic acid is likely DNA
- U indicated the nucleic acid is likely RNA
phosphoesters, phosphodiesters, phosphoanhydrides
- phosphoesters: (phosphate esters), link carbons to phosphate groups
- phosphodiesters: link two different carbons to one phosphate group
- phosphoanhydrides: link two phosphates to each other (within a phosphate group)
describe and compare the primary structure of RNA and DNA
Both: have a net (-) charge, a polar backbone, a phosphodiester linkage following from the 5’ end of the nucleotide to the 3’ end, and a free 3’ -OH group at the 3’ end
RNA: contains uracil instead of thymine and is a little more polar due to the extra -OH on the 2’ carbon of the nucleotide sugars (ribose)
DNA: contains thymine instead of uracil, slightly less polar than RNA due to H+ on 2’ carbon
explain why RNA is susceptible to alkaline hydrolysis whereas DNA is resistant
phosphodiester bond spontaneously hydrolyze (phosphodiesterase) at pH>10
In DNA, the absence of a 2’ OH prevents the reaction. This makes DNA more stable than RBA under alkaline conditions
Nomenclature for polynucleotides
- mononucleotide - 1 nucleotide (ATP, ADP, FMN)
- dinucleotide - 2 nucleotides (FAD, NAD+, dCdG)
- Tri-/Tetra-, etc
- oligonucleotide (more than 20 <50 nucleotides)
- polynucleotides (large number)
Explain why the bases in a nucleic acid “stack: and identify the forces that favour stacking
State Chargaff’s rules and explain what they tell you about a nucleic acid
- nucleotides in DNA were not all
present in equal numbers and base composition varies from organism to organism - Chargaff noted that the amount of A is equal to the amount of T, the amount of C is equal to the amount of G, and the total amount of A/G is equal to the total amount of C/T.
- Chargaff’s “rules” could be satisfied by a molecule with two polynucleotide strands in which A and C in one strand pair with T and G in the other
describe the double helical structure of B-DNA (6)
B-form is the most common form of DNA
- double helix: 2 chains connected to the other through H-bonds between bases (non-covalent), the strands are anti-parallel, with an overall right-handed twist
- hydrophobic core/polar exterior - base pairs largely excluded from water (stabilizes H-bonds) and stack on top of each other - base pair edges are exposed in the major/minor grooves
- The twisting of the DNA “ladder” into a helix creates two grooves of unequal width, the major and minor grooves.
- ribose/deoxyribose and phosphates (very polar) exposed to water
- H-bonds in pairing interactions
- ~10 base pairs/turn
antiparallel: complenatary strands should run in opposite directions of 5’-3’
State how strands of DNA align with one another specifically rather than randomly
hydrogen bonding between the four bases: amino and carbonyl functional groups found in DNA (along with the right nitrogen atoms) allow for specific hydrogen bonding interactions between the bases.
- 2 hydrogen bonds link A/T, T/A
- 3 hydrogen bonds link G/C, C/G
formation of base pairs requires the correct hydrogen bonding interactions between bases in the two strands
Describe how the structure of double-stranded DNA is stabilized
- Hydrogen bonds - between base pairs
- Base stacking (primary stabilising force) - primarily van der Waals forces, hydrophobic forces
State why it is important that the structure of DNA is stabilized by non-covalent forces only
The polar sugar/phosphate backbone is found on the outside while the more hydrophobic bases stack in the core of the structure
State the role for the grooves in the structure of double-stranded DNA
- Other hydrogen bonding sites on the bases are open to the major and minor grooves where they can interact with solvent
List the major differences between RNA and DNA 3-D structures
DNA - double stranded (helical), base pairs A/T, G/C, interstrand base pairing
RNA - single stranded, base pairs A/U, G/C, intrastrand base pairing, partially SS and partially duplex - much more dependant on nucleotide sequence than dsDNA
State why the denaturation of a double helix is feasible and identify the chemical conditions that can cause denaturation
- denaturation must happen for replication or transcription
- Increasing temperature
Define “melting” of a double stranded molecule of DNA
page 28
- separation of the two paired strands (dd→ss)
- disruption of non-covalent forces (base-stacking/hydrogen bonding)
- cooperative process: SLOW & hard to start, easy to continue (FAST zippering)
- changes in absorption properties occur (increase in absorption at 260nm as strands separate
- essential for some cellular processes (replication/transcription)
Define the terms hyperchromicity and hypochromicity with respect to double-stranded nucleic acids
hyperchromicity - a relatively high absorbance, DS→SS DNA
hypochromicity - low absorbance, SS→DS DNA
Explain the term renaturation
- Reformation of DS DNA so it regains its native conformation
- occurs in slow cooling (20-25℃ below Tₘ) - rapid coolin can result in improper base pairing
1. proper base pairing
2. cooperative: nucleation (slow), zippering (fast)
what is Tₘ?
Tₘ - the midpoint of melting
affected by sequence of nucleotides, composition, and solvent
Describe and explain the relationship between the Tm of a double-stranded nucleic acid and its relative content of C and G
Tₘ is higher for for nucleic acids containing more C/G pairs
* base stacking is stronger in G/C pairs: makes 3 hydrogen bonds → G donates 2 and accepts 1, C accepts 2 and donates 1
* A/G pairs only have 2 hydrogen bonds between them (regions of A/T pairs in DNA tend to denature first)
State how and why the Tm of a nucleic acid is affected by pH and ionic strength
slide 31
pH: increasing/decreasing pH will decrease Tm by affecting protonation state
- Tm is highest at pH 7
salt concentrations: ions shield negative charges on phosphate backbone (Mg2+, positively charged proteins)
- low salt → decreases Tm (destabilizes dbl helix)
- high salt → increases Tm (stabilizes dbl helix)
State whether or not RNA can “melt” and justify your answer
RNA can melt but it’s ,=melting curve is less steep and starts at a higher absorbance due to having a mix of SS and DS RNA
- A260nm of free nucleotides > SS > DS
- Abs increases as disorder of structure increases
