8: Nucleotides and Nucleic Acids

Describe three types of RNA

mRNA: messenger RNA; carries protein information in the form of codons to the ribosome to be translated.
tRNA: transfer RNA; key functional component of protein synthesis, translates DNA code into protein code
rRNA: ribosomal RNA; site of protein synthesis

What are four functions of nucleotides/nucleic acids?

1. encoding information (DNA)

2. chemical messaging (mRNA)

3. cofactors/metabolic intermediates

4. energy currency (ATP)

What is the term for a pentose sugar in its closed-ring conformation?

This closed-ring ____ (is/isn't) planar.

β-furanose
It is not planar, it is puckered.

What are the components of a nucleotide?

1. Pentose sugar (ribose or deoxyribose)

2. Nucleotide base

3. 1 or more phosphate groups

What are the components of a nucleoside?

1. Pentose sugar (ribose or deoxyribose)

2. Nucleotide base

What type of bond joins a sugar to a nucleotide base?

At what positions on the sugar and base do this bond occur?

N-β glycosidic bond

The bond is positioned at the 1' position of the sugar.
On the nucleotide base, the bond occurs at the 5-membered ring's NH group.

How are the carbons numbered within a nucleotide?

1' carbon does NOT denote the carbon to which the base is attached (Holmstrom REALLY wants us not to think about it like that.) 1' carbon follows the 1' carbon of a Fischer projection sugar, containing an aldehyde group. The =O is converted to an -OH group, and hemiacetal synthesis causes ring closure, to which the 1' carbon has an attached OH as well as an ester bond.

Carbon labelling just follows around the ring clockwise.

Describe key positions of a nucleotide where key components attach.

Nucleotide bases bind to 1' carbon through a dehydration synthesis reaction; the sugar has an OH group on the 1' position and the base has an H that are both abstracted to form the n-β glycosidic bond.

The phosphate group(s) are typically found bound to the 5' carbon (remember, the 5' carbon is not directly part of the ring) and the 3' carbon. This is what makes up the classic phosphodiester bonds of a nucleotide backbone.

Draw the phosphate backbone of linked nucleotides. Label key carbon positions on the sugar.

What is the difference between the sugars ribose and deoxyribose?

Ribose has hydroxyl groups at both the 2' and 3' positions.
Deoxyribose does not have a hydroxyl group at the 2' position.

DNA is ______ (more/less) susceptible to degradation than RNA

MUCH less.

The lack of a 2' hydroxyl makes DNA more stable and nonreactive than RNA. Further, DNA is typically found in double-stranded form with many repair mechanisms.

How does RNA degradation occur?

RNA's 2' hydroxyl group acts as a nucleophile.
There is a phosphate group at the 3' position, a strong electrophile.
This causes nucleophilic attack that breaks its phosphodiester backbone.

What is the difference between a purine and pyrimidine? Which nucleotide bases are which?

A purine is a 2-ringed structure comprised of 6 member and 5 member nitrogenous rings.

A pyrimidine can be though of as just the 6 membered portion of a purine.

Purines: adenine and guanine
Pyrimidines: thymine, cytosine, uracil.

The purine and pyrimidine bases are ________ (hydrophobic/hydrophilic) at the near-neutral pH of the cell. At acidic or alkaline pH, the bases become _______ (charged/uncharged) and their solubility in water _________ (increases/decreases).

hydrophobic, charged, increases

Think about the sugar phosphate backbone. It's the backbone of DNA because it's the exterior; that is, the charged sugar phosphates have the closest proximity to cellular water. The nucleotide bases are hiding inside because they hate water.

What interactions are involved with base-stacking?

van der Waals interactions, dipole-dipole interactions, hydrophobic effect

Hydrogen bonding isn't really considered ~part~ of base stacking, but it is what contributes to base ~pairing~ in double stranded forms of nucleic acids.

Base pairing = opposite/complementary bases joining via H-bonding
Base stacking = adjacent/same-strand base interactions.

Within base pairing, How many hydrogen bonds between G and C?
How many hydrogen bonds between A and T?

G≡C
A=T

What does primary nucleic acid structure refer to?

Its covalent sequential structure; sequence of nucleotides.
E.g. 5'-GAATCGATCGATTCGACGTA-3'

What does secondary nucleic acid structure refer to?

Any regular, stable structure that nucleotides in a given segment of DNA take.
E.g. represented by a double stranded illustration depicting key features such as hairpins, cruciforms, loops, etc.

What does tertiary nucleic acid structure refer to?

The complex folding patterns of large chromosomes (like in eukaryotic chromatin or tRNA/rRNA)
E.g. helices

DNA's double helix is ______ (left/right) handed. It is very _____ (flexible/rigid).

Right handed helix.
Very flexible structure; rotation is possible about the n-β glycosidyl bond, about each ester bond within the phosphate backbond, as well as slight rotation between 3' and 4' carbon of the sugar.

What factors contribute to the different 3D structures of DNA? (In other words, why is DNA so flexible?)

1. Different conformations of the deoxyribose sugar (endo/exo)

2. Rotation of phosphodeoxyribose bonds (both of the 5' carbon bonds are flexible)

3. Rotation about phosphodiester bonds between attached phosphate groups.

4. Free rotation about the n-β glycosidic bond.

With respect to deoxyribose,

-What conformation(s) are purines able to assume?

-What conformation(s) are pyrimidines able to assume?

Purines can be found either SYN or ANTI to its sugar.

Pyrimidines will be found only ANTI to its sugar; this is due to steric interference by the sugar's C-2 carbonyl oxygen.

Deoxyribose sugars typically take the _____ (endo/exo) conformation

Endo

In general, what are the major/minor grooves in DNA?

DNA helices have two surfaces; the grooves represent the varying distances between the backbones within the helix, due to folding patterns. Changing folding patterns can alter the sizes of the major and minor grooves.

At biological conditions, one helical turn of DNA is how many angstroms long? How many base pairs per turn?

One helical turn is 36 Å. 10.5bp per turn.

How many angstroms apart are nucleotides found in a helix?

Nucleotides are found 3.4Å apart within a DNA helix.

Describe B-form DNA

Right handed helix. The most stable physiological form of DNA. It is the standard reference point that the other forms deviate from.

Describe A-form DNA; helical handedness, general shape, grooviness

Right handed helix. Has a wider helix than B-form, and more base pairs per turn. Essentially, it's like squished/compressed B-form DNA.
It is found in low-water environments. Upon crystallization of DNA, A-form becomes dominant.

Deeper major groove, shallower minor groove.

Describe Z-form DNA; helical handedness, general shape, grooviness

Z is for zig-zag. LEFT-HANDED helix. Essentially, it's like stretched-out B-form DNA.

The glycosyl bond between purines is syn, while for pyrimidines it's anti; this is what causes its left-handedness.

Major groove is barely apparent, minor groove is narrow and deep.

What form does double-stranded RNA typically assume?

What forms is it able and unable to assume?

double stranded RNA typically is found in A-form, however it has been created/observed in Z form.

B-form dsRNA has never been observed.

Order the strengths of the following base-stacking interactions:

Purine-purine
Pyrimidine-pyrimidine
Purine-pyrimidine

In order of strongest to weakest,

purine-purine >> pyrimidine-purine > pyrimidine-pyrimidine

What contributes more strength to base-stacking, A=T or G≡C?

G≡C

Describe the difference between palindromic and mirror repeat sequences.

Palindromic DNA sequences have ~inverted~ repeats; that is, the palindrome center is where it switches strands.

Eg: 5' - T T A G C G C T A A - 3'
3' - A A T C G C G A T T - 5'

Mirror repeats are palindromic sequences on just one strand.

Eg: 5' - T T A G C C G A T T - 3'
3' - A A T C G G C T A A - 5'

What conformations may palindromic and mirror repeat sequences form?

Palindromic sequences may form hairpins/cruciforms.

Mirror repeats cannot form these.

How may a DNA double helix form a triplex?

Watson-Crick (standard) base pairs may pair with a third residue, especially on the major groove. This is known as Hogsteen triplex forming base pairs.

Hogsteen base pairing is most stable at ___ (low/high) pH. Why?

Low pH. It requires the third base to be protonated in order to form a triplex.

How may a DNA double helix form a tetraplex? Is it stable?

If 4 DNA strands have high proportions of guanosine residues, they may form a guanosine tetraplex. It is very stable.

Describe monocystrionic RNA vs polycistrionic RNA.

Monocistrionic RNA codes (has a gene) for one peptide only.
Polycistrionic RNA codes (has a gene) for many peptides.

In eukaryotes, most RNA is polycistrionic. Think about introns, noncoding regions, and alternative splicing.

The product of DNA transcription is always:

A right-handed, helical, single-stranded RNA.

What factors denature DNA? How do these factors affect interactions holding DNA together?

Extremes of heat and/or pH cause disruption to hydrogen bonds that contribute to base-stacking and base-pairing. While no covalent bonds are broken, the DNA unwinds into two strands.

What happens after reversing heat or pH extremes back to biological conditions?

Spontaneous annealing (for partially degraded sequences)
Two step-annealing (for fully degraded strands)
Two step-annealing involves the first, slow process of random collisions and base pairing, then rapid zipping up of the helix.

What is the hypochromic effect?
What is the hyperchromic effect?

Hypochromic effect: UV absorption of a DNA sequences DECREASES as base-pairing INCREASES.
In other words, as strands pair, UV absorption decreases.

Hyperchromic effect: UV absorption of DNA sequences INCREASES as base-pairing DECREASES.
In other words, as DNA is degraded, UV absorption increases.

UV absorption at what wavelength is monitored to determine degradation status of a DNA sequence?

260nm, or 2.60 x 10^-9 m

Why does every unique DNA strand have a unique temperature of degradation (melting point)?

The higher G≡C content, the higher its melting point, because G≡C pairs contribute greatly to holding the strand together under extreme conditions.

During DNA replication and transcription, what sequences are more often seen as the site of unzipping/unravelling (start of replication/transcription)?

Regions with a high proportion of A=T base pairs; because they don't hold onto each other as strong as G≡C.

Rank the following in order of increasing resistance to thermal degradation:

RNA-RNA duplexes
DNA-DNA duplexes
RNA-DNA duplexes

DNA-DNA < RNA-DNA < RNA-RNA

It is unknown why RNA has higher thermal resistance, but it is found to degrade at about 20 C higher temps than DNA.

Why does DNA contain thymine instead of uracil?

Cytosine is deaminated spontaneously into uracil; if DNA normally contained uracil, then it would not be able to recognize mutated U nucleotides from native U nucleotides.

Having thymine acts as a guard that helps DNA repair mechanisms work; all uracil bases are recognized as degradations and thereby repaired.

What are 3 methods of non-enzymatic alterations of DNA?
What do each of these entail?

Deamination, hydrolysis of n-β glycosyl bond (bond between sugar and base)
Deamination: a base loses its exocyclic amino group.
Hydrolysis: Creating of an abasic site (the sugar loses its base)
Radiation: Ring creation and ring opening, breaking of DNA's covalent backbone.

In DNA, hydrolysis of n-β glycosyl bond occurs more frequently in ______ (purines/pyrimidines)

Purines.

What factors accelerate hydrolysis of n-β glycosyl bonds in DNA?

Addition of dilute acid.

What three types of factors contribute to accelerating DNA damage?

1. deaminating agents (nitrous acids/nitrites) cause loss of a base's exocyclic amino group

2. alkylating agents cause modification of bases that disrupt base pairing and base stacking interactions 3)***Oxidative damage: oxidation of deoxyribose/caused by reactive oxygen species: hydroxyl radicals are the most prevalent effectors.

Name two functions of DNA methylation within E. coli

1. Defense: E. coli methylates its own genome to distinguish against foreign DNA

2. Distinction between old/new DNA: the longer DNA exists, the more methylated it becomes. This allows E. coli to recognize younger vs. older DNA within its genome.

Adenine and cytosine are _____ (more often/less often) methylated than guanine and thymine.

Adenine and cytosine are MORE OFTEN methylated than guanine and thymine.

What is the purpose of PCR?

To replicate and amplify a given DNA sequence.

What chemical components are required for PCR? What do each of them do?

DNA Polymerase: Catalyzes the synthesis of DNA
dNTPs: nucleotide bases that are used for DNA synthesis
Template DNA: the strand that is replicated by DNA polymerase
Two primers flanking the target sequence: allow targeted replication

What are the steps of PCR?

1. Add all components to DNA soup

2. Heat reaction to denature and separate target DNA strands

3. Cool reaction to allow primers to bind to DNA template

4. Using available dNTPs, Taq (DNA) polymerase catalyzes synthesis of target sequence

5. Repeat until desired amplification is achieved.

What is the goal of Sanger-sequencing?

To determine the sequence of an unknown segment of DNA via the usage of unlabeled dNTPs in conjunction with fluorescently labeled ddNTPs.

What chemical components does Sanger-sequencing require?

Similar to PCR, Sanger-sequencing requires:
DNA-Polymerase
DNA primers
Excess, unlabeled dNTPS
Fluorescently labeled ddNTPs.

What is the role of ddNTPs in Sanger-sequencing?

ddNTPs (dideoxynucleotide phosphates) are randomly selected along with dNTPs during the process of strand elongation, by the DNA polymerase responsible for synthesis.

However, because ddNTPs lack a 3' hydroxyl, the strand to which it was added is unable to elongate further.

ddNTPs halt chain elongation, and because they are fluorescently labelled for each nucleotide, it can be determined what nucleotide is present at a given position in a DNA strand.

Describe reversible-terminator sequencing.

RT-sequencing uses lasers to excite fluorescently labelled nucleotides. The nucleotides are added one by one, and contain blocking groups that, when added to a sequence via DNA polymerase, only allow one nucleotide to be added to a strand. This allows locations of each nucleotide to be determined in a given sequence.

Describe single-molecule real-time sequencing.

SMRT sequencing utilizes special cells with pores smaller than the wavelength of visible light. A single DNA polymerase is fixed to each pore. Genomic DNA is sheared, and oligonucleotide hairpin sequences within it are ligated to each end of the DNA fragments, generating a circular loop.

Polymerase is added to the end of the single strand of DNA via the usage of primers, initiating synthesis.

Using pulses of light generated by the addition of fluorescently tagged nucleotides, a detector can record the sequence that is synthesized within the circular loop of DNA.

Describe the error rate of SMRT-sequencing and how it is mitigated.

SMRT-sequencing has a high error rate; however, because synthesis occurs around a circular portion of DNA, the elongation (and thus reading/sequencing) process can essentially happen multiple times, so the detector software can account for possible errors and correct them over time as it becomes more certain.