Unit II- Introduction to Nucleic Acids Flashcards
Central dogma of molecular biology
DNA => RNA => Protein
- genetic info stored in DNA
- Double stranded DNA is genetic material
- most common form of DNA is double stranded molecule composed of two-antiparallel strands linked together through hydrogen bonds by complementary bases (Chargaff’s rule)
- sense strand carries the coded genetic information
- antisense strand consists of a complementary sequence of bases oriented in the opposite direction
- genetic info transcribed from DNA to RNA, with the antisense strand of DNA serving as a template except uracil replaces thymine
- genetic info is translated from mRNA to amino acids with redundant nucleotide triplet code
Genome
- most organisms have a double stranded DNA genome
- Virsuses can have dsDNA, ssDNA, dsRNA, or ssRNA genomes:
- dsDNA- Herpes, Smallpox, Papilloma
- ssDNA- Bacteriophage, Parvovirus
- dsRNA- Rotavirus
- ssRNA +sense- Hepatitis C, Dengue, Rubella; -sense: Measles, Mumps, Influenza
Structural components of nucleic acids
- nucleic acids are a chain of polymer composed of a unit called nucleotide
- a nucleotide consists of a sugar, one to three phosphates and a base
- nucleotides are linked together by covalent phosphodiester bonds
- the principle different between the DNA and RNA is that the sugar in DNA lacks a 2’ hydroxyl group
- four kinds of bases: adnenine (A), cytosine (C), guanine (G), and thymine (T)
Nucleotides/ Nucleoside
-building blocks of nucleic acids (both DNA and RNA) contain a sugar ring, a nitrogenous base, and one to three phosphate groups
- Base + Sugar + Phosphate = Nucleotide
- Base + Sugar = Nucleoside
Sugars
- ribonucleic acid (RNA) contains ribose, a five-ring (pentose) sugar
- deoxyribonucleic acid (DNA) contains 2-deoxyribosome, where the hydroxyl (-OH) group at Carbon-2 position is replaced with a hydrogen (-H) group through hydrolysis
Phophates
-up to three phosphate groups can be added to a nucleoside to form nucleotide mono-di-, or tri-phosphate, abbreviated as NMP, NDP, NTP
Sugar-phosphate linkage
The phosphate groups form phosphodiester linkages with the 5’-hydroxyl and the 3’ hydroxyl groups on the sugar to link the building blocks of nucleic acids into a polymeric chain
-the polymeric chains has the 5’ to 3’ carbon positions on the sugar molecule directionality
Bases: Pyrimidine
- Cytosine, Thymine, and Uracil
- contain a flat planar 6-member ring with two nitrogens. The bond between pyrimidines and the sugar-phosphate is between position 1 on the pentose sugar to position 1 (-N) in the pyrimidine
- bases can be mutated or substituted in both DNA and RNA molecules
- cytosine can become uracil through deamination by losing an amino (NH2) group
- thymine can become uracil through demethylation by losing a methyl (Ch3) group
Bases: Purine
- adenine and guanine
- contain a flat planar 6- member ring fused to a 5-member ring, with two nitrogens in each
- the bond between the purines and the sugar-phosphate is from position 1 on the pentose sugar position 9 (-N) of the purine
Nomenclature of bases and nucleotides
Base: Adenine, Guanine, Cytosine, Uracil, Thymine
Nucleoside: Adenosine, Guanosine, Cytidine, Uridine, Thymidine
Nucleotide monophosphate: Adneylate, Guanylate, Cytidylate, Uridylate, Thymidylate
- Nucleoside (name) diphosphate
- Nucleotide (name) triphosphate
Base modifications
-can occur naturally or a form of DNA damage, some natural modifications are important for epigenetic control
Modified bases in DNA:
- 5 methyl cytosine (influences packaging of chromosomal DNA, important for X-chromosome inactivation)
- 5-hydroxymethylcytosine (may regulate gene expression by inducing DNA demethylation, found at high level in the CNS)
Modified bases in RNA:
- hypoxanthine (found in the anticodon of tRNA, also used in purine biosynthesis)
- pseudouracil (found in tRNAs)
- N6-methyladenosine (found in mRNAs and may affect gene expression and splicing
Nucleotide synthesis
- in de novo synthesis, the base itself is synthesized from simpler starting materials, including amino acids. ATP hydrolysis is required
- in salvage pathways, a base is reattached to a ribose, in an activated form, PRPP.
- both de novo and salvage pathways lead to the synthesis of ribonucleotides, consistent with the notion that RNA proceded DNA in the course of evolution
Nucleotide de novo synthesis
- pyrimidines is easier to make
- starts from orotic acid and a sugar is added to make UMP which then splits to CMP and TMP
- the framework for pyridimine base is assembled first and then attached to ribose
- in contrast, the framework for a purine base is synthesized piece by piece directly onto a ribose-based structure
- sugar starts and the purine ring synthesis is added to make IMP which then splits to AMP and GMP
Purine de novo biosynthesis
- Formation of PRPP: De movo purine biosynthesis, like pyrimidine biosynthesis, requires PRPP, an activated ribose intermediate. PRPP requirements for purine and pyrimidine biosynthesis are, however, different. For purine, PRPP provides the foundation on which the bases are constructed sequentially
- Formation of the Purine Ring: All purine nitrogens come from amino acids (glutamine, aspartate, and glycine). Purine nucleotide biosynthesis is a complex of 10 steps process that leads to the formation of IMP (Inosine monophosphate, or Inosinate)
- Formation of AMP and GMP: IMP is a branch point that leads to AMP or GMP. Conversion of IMP to MGP requires ATP, and the first step is feedback-inhibited by GMP. Conversion of IMP to AMP requires GTP and first step is feedback inhibited by AMP. Each nucleotide is inhibited by the end product of each pathway
Conversion to Di- and Tri-Phosphates:
-the nucleoside monophosphates are readily converted into di- and tri-phosphates through kinase activities. Reactions require ATP as donor of phosphates
Conversion of NTPS to dNTPS- AMP, GMP, UMP, CMP are reduced to deoxyribonucleotides for DNA synthesis. Before they can be reduced they must all be converted to nucleoside diphopshates by thioredoxin reductase, ribonucleotide reductase, and thioredoxin
10-Formyl-Tetrahydrofolate
- two steps in purine de novo biosynthesis require 10-formyl-tetrahydrofolate
- one product is tetrahydrofolate which then regenerates
- tetrahydrofolate can be depleted by the action of thymidylate synthesis in the synthesis of dTMP from dUMP
- unless tetrahydrofolate is regenerated from dihydrofolate by dihydrofolate reductase (DHFR) de novo purine and pyrimidine biosynthesis are both blocked
- Cancer cells consume dTMP at a high rate
- therefore the enzyme that catalyzes the conversion of dUMP to dTMP can be inhibited by flurodeoxyuridylate
- enzyme DHFR can also be inhibted by antifolate drugs
Purine salvage pathway
- free purine bases can be attached to PRPP to form purine nucleoside monophosphates, in a reaction analogous to the formation of orotidylate two salvage enzymes with different specificities recover purine bases
- adenine phosphoribosyltransferase catalyzes the formation of adenylate
- Adenine + PRPP –> adenylate +PPi (pyrophosphate)
- hypoxanthine-guagine phosphoribosyltransferase catalyzes the formation of guanylate as well as inosinate
- Guanine +PRPP –> guanylate + PPi
- Hypoxanthine + PRPP –> inosinate + PPi
Nucleic acid catabolism
- nucleobases (thymine, guanine, etc) and nucleoside monophosphates can be interconverted by action of the enzyme phosphoribosyl transferase in the presence of PRPP
- mononucleotides can be converted to nucleoside tiphospates and DNA, or can be converted to either nucleosides or broken down to nucleobases
- all purine degradation leads to uric acid and excreted into urine as insoluble crystals. It can be further broken down to allantoin, allotoic acid, urea and ammonia
- ingested nucleic acids are degrdaded to nucleotides by pancreatic nucleases, and intestinal phosphodiesterases in the intestine
Pathways of purine metabolism in humans
- deficiencies in the listed enzymes have all been linked to human diseases that are often neurological disorders
- disorders have been linked to defects not only in purine metabolism, but also in pyrimidine metabolism
- Adenosine deaminase (ADA); Adenine phosphoribosyltransferase (APRT); Hypoxanthine-guanine phosphoribosyltransferase (HPRT); nucleoside phosphorylase (NP); 5’-nucleotidase (NT, 5’); PRPP amidotransferase (PAT); phosphoribosylpyrophosphate (PRPP); PRPP synthetase (PRPPS); xanthine oxidase (XO)
Clinical relevance of nucleotide synthesis and metabolism
Gout:
- defects in PRPP synthetase and HGPRT (hypoxanthine-guanine phosphoribodyltransferase)
- impaired excretion or overproduction of uric acid
- uric acid crystals precipitate into joints (Gouty Arthritis), kidneys, ureters (stones)
- lead impairs uric acid excretion-lead poisoning from pewter drinking goblets
- xanthine oxidase inhibitors inhibit production of uric acid, and treat gout
- allopurinol treatment- hypoxanthine analog that binds tp Xanthine Oxidase to decrease uric acid production
Lesch-Nyhan syndrome
- rare inherited disorder caused by deficiency of the enzyme HGPRT, X-linked
- causes increased level of hypoxanthine and guanine (lead to increased degradation to uric acid)
- also causes accumulation of PRPP and stimulates production of purine nucleotides (and thereby increases their degradation)
- causes gout-like symptoms, but also neurological symptoms: spasticity, aggressiveness, self-mutilation
- first neuropsychiactric abnormality that was attributed to a single enzyme
Cellular functions of nucelotides
- building blocks of nucleic acid polymers, DNA and RNA
- energy carriers (ATP,GTP)
- important components of coenzymes: FAD, NAD(P)+ and coenzyme A
- precursors for second messengers: cAMP, cGMP
- activated intermediates in many biosynthetic pathways: e.g. S-adenosylmethionine (SAM) as methyl donor
Bonds and base pairing
- antiparallel strands and complementary base pairing
- 2 hydrogen bonds for AT
- 3 for GC
The double helix: B-DNA conformation
- right handed helix
- the plane of the base is perpendicular to the S-P backbone
- one turn of the helix equals 10.5 base pairs 34 A
- the twisted of the helix forms major and minor grooves
Other forms of the double helix
A-DNA:
-right,handed, repeating unit 1 bp, bp/turn = 11, length/turn= 28 A, dehydrated DNA
B-DNA:
-right handed; repeating unit 1 bp; bp/turn = 10.5; length/turn = 34 A
Z-DNA:
-left handed; repeating unit 2 bp; bp/turn= 12; length/turn 45 A, negative supercoiling or high salt
Conformational changes of the B-DNA: DNA bending
- DNA bending- certain sequences can cause the double helix to bend, forming a larger major groove and a smaller groove
- DNA binding proteins reach into the grooves of DNA to make contact with side-groups of specific base pairs
- each individual base pair deviates significantly from the average B-DNA conformation in a manner that depends on the surrounding sequence of bases
- this forms another possible basis for how DNA binding proteins can recognize specific bases out of the otherwise very regular DNA structure. The binding of these proteins can in turn alter the structure of DNA, causing it to take on a form that facilitates the binding of other proteins
Covalent Modifications- base modifications such as the aforementioned N6-methyl-deoxyadenosine or the 5-methyl-deoxycytidine can affect the structure of DNA but can also directly affect the binding of proteins
Hoogsteen base pairing
- the purine bases could flip from their normal anti-conformation to a syn conformation and form a different set of hydrogen bonds with their pyrimidine partners
- transient Hoogsteen base pairs exist in canonical duplex DNA
Denaturation of DNA
Tm: melting temperature, the temperature at which half of the double-stranded DNA molecules dissociate into single-stranded DNA
- determined by size
- GC content ( more GC pairs imply more H bonds to be broken and requirement for higher input of energy)
- Ionic strength- Tm is proportional to salt concentration, high salt concentrations stabilize double stranded DNA by binding to negatively charged phosphates in backbone and decreasing repulsion between chains
- pH- Tm is inversely proportional to the pH, high concentrations of OH form hydrogen bonds
- other reagents that can form hydrogen bonds with bases decrease Tm (formamide)
Renaturing of DNA
- denatured DNA can be renatured and eventually reform the correct hydrogen bonds and the original double helical structure
- slow cooling allows complementary sequences
- single-stranded DNA bound to a surface such as nylon or a glass microchip, can renature with complementary DNA or RNA sequence called hybridization
- high stringency- At or close to the Tm- only perfect matches can form
- low stringency- below the Tm- under conditions that stabilize the double helix (high salt or low temperature) - imperfect base pairing
Chromosome topological stress
- a circular DNA molecule isolated in nature is usually a superhelix with a Watson-Crick double helix crossing over itself forming supercoils
- relaxed form as shown contains nicks in the double strand
- negative supercoils are found in underwound DNA wherease positive supercoils are found in overwound DNA
- Type I- cuts one strand
- Type II- cuts both strands
Structural features of RNA (vs. DNA)
- single stranded
- shorter in length
- complex tertiary structure
- unstable: vulnerable to base- catalyzed hydrolysis
- RNA can also form intramolecular hydrogen-bonded base pairs resulting in secondary structures such as hairpin and stemloop-important for function and recognition
Messenger RNA
- 5% of total RNA, most hetergeneous in size of the RNA types
- contain genetic information copied from specific regions of DNA to be used as a template for protein synthesis
Non-coding RNAs
-ncRNAs
including microRNAs (miRNAs)
Ribozymes etc
Ribosomal RNA (rRNA)
- 80% of total RNA
- consists of several species of distinct sizes that are part of the structure of the ribosome (ribosomes are the subcellular structures where protein synthesis occurs)
Transfer RNA (tRNA)
- 15% of total RNA
- small RNA molecules (composed of 73-93 nucleotides)
- contain elaborate secondary structures and some unique nucleotides
- serve as “adaptor molecules” in protein synthesis- recognize the code in the messenger RNA indicating which amino acid comes next in a protein and brings that amino acid to the site of the protein synthesis on the ribosome. Have at least one specific tRNA molecule for each of the twenty amino acids
miRNAs
- small endogenous RNAs of ~22 nt that play important regulatory roles in animal development
- bind to complementary sites of specific mRNAs to inhibit their translation
Small interfering RNA (siRNA)
- 20-25 bp double stranded RNA molecules
- function in RNA interference (RNAi) pathway
Ribozymes
-have elaborate secondary structure, which can form an active site that can catalyze intramolecular reactions and reactions with other RNA molecules much in the same way as enzymes
Types of RNA in the cell
1) Messenger RNA (mRNA)
Contain genetic information copied from specific regions of DNA to be used as a template for protein synthesis
2) Non-coding RNAs (ncRNAs)
- Ribosomal RNA (rRNA): part of the ribosome
- Transfer RNA (tRNA): adaptor molecule between mRNA and amino acids during protein synthesis
- snoRNA, miRNA, siRNA, snRNA, exRNA, piRNA, and long ncRNAs
- Ribozymes: catalytic RNA molecules
