Nucleotides and Nucleic Acids
Chapter 8 Nucleotides & Nucleic Acids
Nucleotides are the building blocks of nucleic acids A nitrogenous base A phosphate group
(pyrimidines or purine)
A pentose sugar
Nucleotides have three characteristic components Chapter 8
Structure of a Nucleotide
We Need Nucleic Acids!
The Ribose Sugar
DNA Protein Protein Trait DNA RNA mRNA RNA
rRNA
Pol
tRNA
3
• DNA contains genes, the information needed to synthesize functional proteins and RNAs – DNA also contains segments that play a role in regulation of gene expression (promoters, operators, etc.) • Messenger RNAs (mRNAs) are transcribed from DNA by RNA polymerases and carry genetic information from a gene to the ribosome complex
• Ribose (β-Dribofuranose) is a pentose sugar • Note numbering of the carbons. • In a nucleoside or nucleotide, "prime" is used (to differentiate from base numbering).
• Ribosomal RNAs (rRNAs) are components of the ribosomes and play a role in protein synthesis in conjunction with the template mRNA and the AA carrying tRNA
5 4
1 3
2
• Transfer RNAs (tRNAs) carry the AAs designated by the codons of the mRNA and bind to the Ribosome to help form the growing polypepide chain Chapter 8
2
Chapter 8
4
1
Structure of a Nucleotide
Structure of a Nucleotide
The Pyrimidine and Purine Bases
Bases from the minor leagues…
Big word, small ring
Small word, big ring
Know these: Numbering System and General Ring Structure Chapter 8
5
• 5-Methylcytidine occurs in DNA of animals and higher plants • N6-methyladenosine occurs in bacterial DNA Chapter 8
These 8 bases you do not need to know…
Structure of a Nucleotide
Structure of a Nucleotide
Major Bases in Nucleic Acids
The Phosphate
7
• The bases are abbreviated by their first letters (A, G, C, T, U). • The purines (A, G) occur in both RNA and DNA
• The pyrimidine C occurs in both RNA and DNA, but • T occurs only in DNA, and U occurs only in RNA Chapter 8
Be able to recognize and draw these five!
A nucleoside + one or more phosphoryl groups is called a nucleotide. 6
Chapter 8
8
2
Structure of a Nucleotide
Structure of a Nucleotide
Linkages
Phosphate Variations: Number
The phosphate at the 5’ carbon Sugar to the base at 1’ carbon
1 2 3
Note the hydroxyl at the 2’ carbon Chapter 8
Why are these important?
Nucleoside mono-, di-, or triphosphate
9
Chapter 8
Structure of a Nucleotide
Structure of a Nucleotide
Nucleotides in Nucleic Acids
Ribonucleotides • The ribose sugar with a base (here, a pyrimidine, uracil or cytosine) attached to the ribose C-1' position is a ribonucleoside (here, uridine or cytidine).
• Bases attach in β-linkage to the C-1' of ribose or deoxyribose • The pyrimidines attach to the pentose via the N-1 position of the pyrimidine ring
• Phosphorylate the 5' position and you have a ribonucleotide (here, uridylate or cytidylate)
• The purines attach through the N-9 position • Some minor bases may have different attachments.
Chapter 8
11
• Ribonucleotides are abbreviated (for example) U, or UMP (uridine monophosphate) 10
Chapter 8
12
3
Structure of a Nucleotide
Structure of a Nucleotide
Deoxyribonucleotides – Better for DNA!
The Major Ribonucleotides
• A 2'-deoxyribose sugar with a base (here, a purine - adenine or guanine) attached to the C1' position is a deoxyribonucleoside • Phosphorylate the 5' position and you have a nucleotide • Deoxyribonucleotides are abbreviated (for example) A, or dA or dAMP
Note the N9 (purine) and N1 (pyrimidine) linkage to the ribose ring at C1’ Chapter 8
13
Chapter 8
15
Structure of a Nucleotide
Structure of a Nucleotide
One of RNA’s Little Problems – Stability!
The Major Deoxyribonucleotides
DNA, which lacks a 2’-OH, is stable under alkaline conditions…
Chapter 8
14
Chapter 8
16
4
Structure of a Nucleotide
Structure of a Nucleotide
Now, on to Nucleic Acids!
Nucleotide Nomenclature
• Nucleotide monomers can be linked together via a phosphodiester linkage formed between the 3' -OH of a nucleotide and the phosphate of the next nucleotide. • Two ends of the resulting poly- or oligonucleotide are defined: – The 5' end lacks a nucleotide at the 5' position
Chapter 8
17
Chapter 8
– The 3' end lacks a nucleotide at the 3' end position 19
Structure of a Nucleotide
Structure of a Nucleotide
Nucleotide Nomenclature
Sugar-Phosphate Backbone Berg et al. “Biochemistry” 5th Ed. Fig. 1.1
• The polynucleotide or nucleic acid backbone thus consists of alternating phosphate and pentose residues. • The bases are analogous to side chains of amino acids; they vary without changing the covalent backbone structure. • Sequence is written from the 5' to 3' end: 5'-ATGCTAGC-3' • Note that the backbone is polyanionic, and the phosphate groups have a pKa ~ 0. Chapter 8
18
Chapter 8
20
5
Structure of a Nucleotide
Nucleic Acid Structure
The bases can take syn- or anti- positions
Polynucleotides vs. Polypeptides • As in proteins, the sequence of side chains (bases in nucleic acids) plays an important role in function. • Nucleic acid structure depends on the sequence of bases and on the type of ribose sugar (ribose, or 2'-deoxyribose). • Hydrogen bonding interactions are especially important in nucleic acids, both interstrand and intrastrand.
Chapter 8
21
Chapter 8
Structure of a Nucleotide
23
Nucleic Acid Structure
Sugar phosphate backbone conformation
DNA
• Polynucleotides have unrestricted rotation about most backbone bones (within limits of sterics) • One exception is the sugar ring bond
Chapter 8
• DNA consists of two helical chains wound around the same axis in a right-handed fashion aligned in an antiparallel fashion.
• This behavior contrasts with the peptide backbone.
• There are 10.5 base pairs, or 36 Å, per turn of the helix.
• Also in contrast with proteins, specific, predictable interactions between bases are often formed: A with T, and G with C.
• Alternating deoxyribose and phosphate groups on the backbone form the outside of the helix.
• These interactions can be interstrand, or intrastrand.
• The planar purine and pyrimidine bases of both strands are stacked inside the helix.
22
Chapter 8
24
6
Nucleic Acid Structure
Nucleic Acid Structure
DNA
Stabilization of Double Helix • Weak forces stabilize the double helix:
• The furanose ring usually is puckered in a C-2' endo conformation in DNA.
(1) Hydrophobic Effects: Burying purine and pyrimidine rings in the interior of the helix excludes them from water
• The offset of the relationship of the base pairs to the strands gives a major and a minor groove.
(2) Stacking interactions: Stacked base pairs form van der Waals contacts (3) Hydrogen Bonds: H-bonding between the base pairs (not on the backbone!)
• In B-form DNA (the most common) the depths of the major and minor grooves are similar to each other.
(4) Charge-charge interactions: Electrostated repulsion of negatively charged phosphate groups is decreased by cations (e.g. Mg2+) and cationic proteins
• The base-pairs are perpendicular to the helical axis Chapter 8
25
Chapter 8
27
Nucleic Acid Structure
Nucleic Acid Structure
DNA Strands
Interstrand H-bonding Between DNA Bases
• The opposing strands of DNA are not identical, but are complementary. • This means: they are positioned to align complementary base pairs: – C with G, and A with T, but the strands run antiparallel to each other
• You can thus predict the sequence of one strand given the sequence of its complementary strand. • Note that sequence conventionally is written from the 5' to 3' end • Such a structure is useful for information storage and transfer! Chapter 8
Watson-Crick base pairing 26
Chapter 8
28
7
Nucleic Acid Structure
Nucleic Acid Structure
Base Stacking in DNA
DNA Structure Summary
• C-G (red) and A-T (blue) base pairs are isosteric (same shape and size), allowing stacking along a helical axis for any sequence.
Chapter 8
• Base pairs stack inside the helix.
Berg et al. “Biochemistry” 5th Ed. Figs. 1.4; 5.13
29
Chapter 8
31
Nucleic Acid Structure
Nucleic Acid Structure
Various Nucleic Acid Structures
Unusual DNA Structures
• B form - The most common conformation for DNA. • A form - common for RNA because of different sugar pucker. Deeper minor groove, shallow major groove
36 base pairs Backbone - blue; Bases- gray
– A form is favored in conditions of low water.
• Some structures seen in DNA are sequence dependent
• Z form - narrow, deep minor groove. Major groove hardly existent. Can form for some DNA sequences; requires alternating syn and anti base configurations. Chapter 8
• These structures can be very important in the binding of proteins to the nucleic acid 30
Chapter 8
32
8
Nucleic Acid Structure
Nucleic Acid Structure
RNA
Large RNAs may be highly structured
• In gene expression, RNA acts as an intermediary between the genetic information of DNA and the production of polypeptides
• The 337 nt long M1 RNA of the tRNA processing enzyme RNase P of E. coli has many hairpin loops • Even for a random RNA sequence, about 60-70% may be involved in secondary structure (why?)
• In eukaryotes, DNA is largely confined to the nucleus whereas protein production occurs in the cytoplasm. • RNA allows these two processes to remain separated by shutting between them • The product of transcription is always a single stranded RNA
G-U pairs
– Tends to assume a right handed helical conformation dominated by base-stacking interactions
G-U pairs are also allowed, as they are energetically neutral.
• Any self-complimentary sequences or interactions with a second RNA or a DNA chain will lead to duplex formation with one difference from duplex DNA: Uracil instead of Thymine Chapter 8
33
Nucleic Acid Structure
Nucleic Acid Structure
RNA has a Rich and Varied Structure
RNA Displays Interesting Tertiary Structure
• Watson-Crick base pairs in helical segments (usually A-form).
Singlestranded RNA right-handed helix
• Helix is secondary structure. Note A-U pairs in RNA
Green = phosphodiester backbone
DNA can also form structures like this.
Chapter 8
Other regions may also pair, which facilitates tertiary folding.
Chapter 8
35
Yeast tRNAPhe (1TRA)
Hammerhead ribozyme (1MME)
T. thermophila intron, A ribozyme (RNA enzyme) (1GRZ) 34
Chapter 8
36
9
Nucleic Acid Chemistry
Nucleic Acid Chemistry
• Replication: The preparation of exact copies of a molecule. DNA replication is essential for life, and is what allows organisms to create copies of themselves. • Transcription: The copying of the genetic message encoded in DNA to RNA • Translation: The translation of the genetic message encoded in RNA into a polypeptide with a specific amino acid sequence
• In DNA replication, each DNA strand serves as a template for the synthesis of a new strand, producing two DNA molecules, each with one new strand and one old strand. • This is termed semiconservative replication.
• Performing these functions requires transformations of these complex molecules to occur without losing or damaging the genetic information (i.e. causing mutations). • Thus, chemical modifications of nucleic acids must be prevented, controlled, monitored, and fixed if needed. Chapter 8
DNA Replication
37
• Replication requires separation of the DNA strands (transient melting, or denaturation) so that the parent strands can serve as templates. Chapter 8
Nucleic Acid Chemistry
Nucleic Acid Chemistry
Transformations of Nucleic Acids
Partially Denatured DNA • DNA strands can be separated (DNA denaturation) by increasing temperature.
• For DNA and RNA to perform their respective and various functions within organisms, they must undergo physical and chemical changes themselves Physical
• Denaturation is a result of disruption of interactions that stabilize DNA structure: Hydrogen-bonding
Denaturation (Melting) Renaturation (Annealing) Shearing
Base stacking • This electron micrograph shows DNA that has been partially denatured with heat, and then fixed.
Chemical
Chapter 8
Replication, synthesis Cleavage Deamination Depurination Crosslinking Alkylation Oxidation
39
• The red arrows point to the single-stranded regions, which appear as “bubbles” in the doublestranded backbone. • Would you expect these regions to be the same or different each time you denature this piece of DNA? Why or why not? • What characteristics would these regions have? 38
Chapter 8
40
10
Nucleic Acid Chemistry
Nucleic Acid Chemistry Annealing: Hybridization
DNA Denaturation and TM
• Experiment: completely denature duplex DNAs from a fly and a human.
• Since DNA denaturation is commonly done by increasing temperature, it is also called DNA melting.
• Mix the denatured samples and keep at ~ 65 ˚C for many hours
• This can be followed by observing the change in the ultraviolet absorption band of DNA.
• Under these conditions, complementary strands will anneal. • Most fly strands anneal with fly; and human with human
• Stacked DNA bases absorb less light than unstacked; this is called hypochromism.
• But some strands of fly and human DNA will anneal to give hybrid duplexes.
• The "melting temperature" Tm is taken as the inflection point of the melting curve. Chapter 8
• This reflects a common evolutionary heritage. 41
Chapter 8
Nucleic Acid Chemistry
Nucleic Acid Chemistry
DNA Melting and Annealing
Nucleic Acids Can Be Chemically Altered
• DNA denaturation is reversible. • The formation of double helical DNA from denatured DNA is called annealing.
Deamination Depurination
• Lowering the temperature below Tm (given the right pH, ionic strength, etc.) will allow annealing.
Crosslinking Alkylation
Tm depends on pH, ionic strength, DNA length, and DNA base composition Chapter 8
What would be different if this were done with human and chimp DNA? 43
Oxidation What will result from this…? 42
Chapter 8
44
11
Nucleic Acid Chemistry
Nucleic Acid Chemistry
Base Deamination
Mutation by Deamination
• Nucleotide bases can undergo spontaneous loss of their exocyclic amino groups (deamination). • Under typical cellular conditions, deamination of cytosine in DNA to uracil occurs in about one of every 107 residues every 24 hours.
Uracil
• A and G deamination occurs at 1/10 of this rate. Can now pair with
Chapter 8
45
Chapter 8
47
Nucleic Acid Chemistry
Nucleic Acid Chemistry
Mutation by Deamination
Thymine in DNA Allows Error Correction! • Why is U present in RNA, and T in DNA?
• Deamination of Cytosine is mutagenic. • When deaminated, cytosine becomes uracil which pairs with A (rather than G) • This “mispairing” causes a G to A substitution in the complementary strand.
• Spontaneous deamination of U, which gives C, is relatively common. • This is potentially mutagenic • The enzyme uracil glycosylase recognizes U in DNA and cleaves the glycosidic bond to remove the base. • Mutant cells lacking uracil glycosylase have a high rate of G-C to A-T base pair mutations.
Uracil Chapter 8
46
Chapter 8
48
12
Nucleic Acid Chemistry
Nucleic Acid Chemistry UV Damage to DNA
Depurination
• Formation of a cyclobutane pyrimidine dimer introduces a bend or kink into the DNA helix
• The N-β-glycosyl bond between the base and the pentose can undergo hydrolysis.
• 50-100 of these lesions occur in each skin cell for every second of sun exposure(!)
• This process is faster for purines than for pyrimidines
• Most of these are recognized and repaired immediately.
• 1/100,000 purines are lost from DNA every 24 hours
• If damage goes uncorrected, permanent mutation may result.
• Depurination of RNA and of ribonucleotides is much slower.
Chapter 8
• CC to TT mutation occurs if a CC dimer is mispaired with two adenine bases. This is often seen in the p53 gene in skin cancers. 49
51
Nucleic Acid Chemistry
Nucleic Acid Chemistry
Crosslinking of Bases
Mutation by Methylation
• Pyrimidine dimers can be formed by exposure to UV light.
• Methylation of the guanine carboxyl oxygen results in the formation of a nucleotide that can only form two hydrogen bonds with its partner
• Most commonly seen for adjacent thymidine residues on the same DNA strand • Cyclobutane, or 6-4-linked dimers, can be formed.
• This methylated gaunine can then be pair with T rather than C
• This causes a kink in the helix • X-rays and gamma rays can cause ring opening and base fragmentation. Chapter 8
Chapter 8
• This is a weaker pair in addition to a mutagenic pairing 50
Chapter 8
52
13
Nucleic Acid Chemistry
Nucleic Acid Chemistry Automated DNA Sequencing
Chemical Alkylating Agents – Harmful to Children and Other Living Things…
• One major improvement in recent years has been the development of automated procedures for fluorescent DNA sequencing (Wilson et al., Genomics, 1990, pg. 626). • These procedures generally use primers or dideoxynucleotides to which are attached fluorophores (chemical groups capable of fluorescing). • During electrophoresis, a monitor detects and records the fluorescence signal as the DNA passes through a fixed point in the gel. • The use of different fluorophores in the four base-specific reactions means that, unlike conventional DNA sequencing, all four reactions can be loaded into a single lane. • The output is in the form of intensity profiles for each of the differently colored fluorophores, but the information is simultaneously stored electronically.
• Nitrogen mustard. First symptoms of exposure: skin, eye and respiratory tract irritation, burns and blistering. • Long-term effects include leukemia. • Used as a treatment of Hodgkin's disease and brain tumors (!) Chapter 8
53
Chapter 8
55 Figure 6.17 from Human Molecular Genetics, 2nd Ed., Strachan et al.
Nucleic Acid Chemistry
Nucleic Acid Chemistry Chemical Synthesis of DNA
DNA Sequencing: Dideoxy Method (A) The Dideoxy method relies on the insertion of ddNTPs into a growing DNA chain, which causes chain termination.
(A) NTP is attached to the silica support (B) DMT protecting group is removed
(B) Purified target DNA is synthesized using a primer specific for the strand to be sequenced.
(C) The incoming NTP is activated and coupled to the immobilized NTP to form a 5’ to 3’ linkage
(C) In four separate reactions, four different ddNTP’s are added into the reaction mixture (in addition to the normal dNTP’s) resulting in the formation of DNA strands of varying lengths.
(D) The dinucleic acid is oxidized to produce the phosphotriester
(D) These strands can be separated using electrophoresis and the sequence read by comparing the lanes.
(E) Steps (B) through (D) are repeated (F) The remaining protecting groups are removed
Sequencing Animation
Chapter 8
(G)The oligonucleotide is separated from the support 54
Chapter 8
Why would synthesis of RNA be more difficult?
56
14
Other Functions of Nucleotides • Building blocks of nucleic acids (RNA, DNA) (analogous to amino acid role in proteins) • Energy currency in cellular metabolism (ATP: adenosine triphosphate) • Allosteric effectors • Structural components of many enzyme cofactors (NAD: nicotinamide adenine dinucleotide) Chapter 8
57
15
