Nucleic Acids Flashcards
1868
-Miescher discovered nuclein from pus
1920
-Griffith discovered transmission of pathogenicity in strep pneumoniae
1944
Avery led team-DNA is genetic material
1952
Hershey and Chase-phage T2 can transmit DNA into bacteria
1951
Chargaff showed base ratios in DNA and RNA
152
Rosalind Franklin collected X-ray diffracted images of DNA molecule
1953
Watson and Crick solved double helix
1953
Crick presented central dogma
1958
Meselson and Stahl showed DNA is semi-conservative
2001
first draft of sequence of human genome
central dogma
- DNA to RNA to protein
- DNA is two antiparallel strands linked together through H bonds
sense strand
carries coded genetic information
antisense strand
complementary sequence of bases oriented in opposite direction
-template for mRNA
genome
- all hereditary material
- can be dsDNA, ssDNA, dsRNA, ssRNA
dsDNA
- herpes, smallpox, papilloma,
- Hep B (retro and uses RNA in replication)
ssDNA
- Bacteriphage, Parvovirus B19
- no DNA repair process- high rate of mutations- may be needed to adapt
dsRNA
-Rotavirus
ssRNA
- plus sense- Hep C, Dengue, Rubella
- minus sense- Measles, Mumps, Influenza
- no repair- higher mutation rate
- HIV- but needs DNA in replication
DNA vs RNA sugar
DNA lacks and OH on carbon 2
nucleoside
-sugar and base, no phosphate
phosphodiester bonds
-between 3’ and 5’ of sugar- strand runs 5-3
Pyrimidines
- smaller
- cytosine, thymine, uracil
- cytosine to uracil loses amine
- thymine to uracil loses methyl
- flat planar 6-membered ring with two nitrogens
- bond to sugar/phosphate is 1-sugar to 1-pyrimidine (bottom N)
Purines
- bigger
- Adenine and Guanine
- flat planar 6-member ring fused to a 5 member ring with two nitrogens in each
- 1-sugar to 9 purine (bottom N on 5 member ring)
Adenine
- nucleoside is Adenosine
- NMP is Adenylate (AMP_
Guanine
- Guanosine
- Guanylate (GMP)
Cytosine
- Cytidine
- CMP-Cytidylate
Uracil
- Uridine
- UMP- Uridylate
Thymine
- Thymidine
- Thymidylate
Nucleotide
- NMP or dNMP
- nucleoside and 1 phosphate
Nucleoside diphosphate
- NDP of dNDP
- nucleoside and 2 phosphates
Nucleoside triphosphate
- NTP or dNTP
- nucleoside and 3 phosphates
- immediate precursors for RNA or DNA synthesis
5-methyl-cytosine
- influences packaging of chromosomal DNA
- important for X chromosome inactivations
5-hydroxylmethylcytosine
-may regulate gene expression by inducing DNA demethylation, found at high level in CNS
Hypoxanthine
- found int 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
- can be de novo or salvage
- de novo-synthesized from simpler starting materials, including amino acids. needs ATP hydrolysis
- salvage-base reattached to ribose in activated form called PRPP
- both pathways lead to synthesis of ribonucleotides first- RNA before DNA in evolution
de novo synthesis of pyrimadines
- orotic acid plus sugar–>UMP–>CMP and TMP
- framework for base first then attached to ribose
de novo purine synthesis 1
- sugar + purine ring synthesis–>IMP (from hypoxanthine) –> AMP and GMP
- sugar first base added piece by piece
de novo purine synthesis 2
- PRPP (activated sugar) provides foundation on which bases on constructed
- ring for purine from 10 step process that leads to formation of IMP. N comes from aa
- IMP is branch point for AMP or GMP
- IMP to GMP needs ATP (inhibited byGMP)
- IMP to AMP needs GTP and aspartic acid (inhibited by AMP)
- nucleoside monophosphate converted to di and tri through kinase activity
- nucleoside diphosphates are reduced to deoxyrobonucleotides
reduction reaction requires
- thioredoxin reductase
- ribonucleotide reductase
- thioredoxin
10-formyl-tetrahydrofolate
- two steps in purine biosynthesis
- one product is tetrahydrofolate- regenerates 10-formyl
- tetrahydrofolate can be depleted by thymidylate synthase in synthesis of dTMP from dUMP, but is regenerated by dyhydrofolate reductase (DHFR) (from dihydrofolate)
- if tetrahydrofolate isn’t regenerated, no de novo synthesis of purines or pyrimidines
- dTMP pathway for cancer therapy because cancer cells consume dTMP-block thymidylate synthase with fluorodroxyuridylate
- DHFR block- no tetrahydrofolate, no synthesis, no growth
**See picture
purine salvage pathways
- pre made bases
- adenine + PRPP—> adenylate (AMP) +PPi-adenine phosphoribosyltransferase
-Guanine + PRPP—> guanylate (GMP) +PPi
-Hypoxanthine + PRPP—> inosinate (IMP) +PPi
^both hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
-can then form NDP, NTP, dNDP, dNTP
nucleic acid catabolism
- bases and NMP can be interconverted by phosphoribosyltransferase in presence of 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRPP)
- mononucleotides (NMPs) can go to NTPs and DNA, nucleosides, or nucleobases (T,C)
- all purine degradation leads to uric acid which is excreted into urine as insoluble crystals
- further breakdown to allantoin, allantoic acid, ammonia
- ingested nucleic acids broken down by pancreatic nucleases and intestinal phosphodiesterases
metabolism pathways in humans
-look at pg 20
ADA
adenosine deaminase
APRT
adenine phosphoribosyltransferase
HPRT
hypoxanthine-guanine phosphoribosyltransferase
NP
nucleoside phosphorylase
5’ NT
5’ nucleotidase
PAT
PRPP amidotransferase
PRPP
phosphoribosylphosphate
PRPPS
PRPP synthetase
XO
xanthin oxidase
gout
- defects in PRPP synthetase and HGPRT
- uric acid crystals precipitate into joints, kidneys, and ureters
- treatment with xanthine oxidase inhibitors
- lead impairs uric acid secretion
Lesch-Nyhan sydrome
- rare inherited disorder
- deficiency of HGPRT
- causes increased level of hypoxanthine and guanine (increased degradation of uric acid)
- causes accumulation of PRPP and stimulates production of purine nucleotides
- causes gout like symptoms, but also neurological symptoms
- first neuropsychiatric abnormality attributed to a single enzyme
cellular functions of nucleotides
- building blocks for nucleic acid polymers, DNA, RNA
- energy carriers
- important components of co-enzymes: FAD, NAD(P)+ and coA
- precursors for second messengers-c/gAMP (cAMP-AMP lose caffeine jolt)
- activated intermediates in many biosynthetic pathways-S-adenosylmethionine (SAM) as methyl donor
Base pairing and H bonds
- 2 for AT and 3 for GC
- antiparallel
B-DNA conformation
- most common
- right handed helix
- plane of base is perpendicular to the SP backbone
- 1 turn is 10.5 bp, 34 angstroms, 3.4 nm
- major and minor grooves
- antiparallel
- repeating unit is 1 bp
A-DNA
- right handed
- repeating unit is 1 bp
- 11 bp per turn, 28 angstroms
- appears in dehydrated samples
additional conformations of DNA
- favored by certain base sequences, salt conditions, base modifications, and humidity
- single helix can contain A, B, and Z conformations
Z-DNA
- left handed
- repeating unit is 2 bp
- 12 bp/turn, 45 angstroms
- not favorable
- alternating purine/pyrimidine, neg supercoiling, high salt can induce Z formation
DNA bending
- facilitates protein-DNA interactions
- larger major groove and smaller minor groove
- proteins interact with side groups of a bp
- each individual bp deviates from B conformation (depending on surrounding bases)-how binding proteins recognize- then alter more for other proteins
- covalent mod-affect structure and binding
hoogsten bp
- purine bases can flip from normal anti conformation to a syn conformation and form different set of H bonds with pyrimidine partners
- 1% of the time these bp exist in canonical duplex DNA
- proteins probably recognize altered structure
- another layer of gene regulation
tm
- melting temp
- half dsDNA molecules dissociate into ssDNA
- determined by size, GC content, salt concentration (high stabilizes), pH, other reagents
- cloning, southern blot, FISH, microarrays, PCR
- increase temp, pH, lower salt to melt DNA
- lower GC content easier to melt
- other reagents that can H bond with single stranded DNA stabilize it and decrease Tm
denatured DNA
- can be renatured and reform correct H bonds
- slow cooling allows complementary sequences to H bond
complementary sequences
-single stranded sequences capable of H bonding with each other
hybridization
- ssDNA bound to nylon or a glass microchip can still renature with complementary strand
- high or low stringency
high stringency
at or close to Tm- only perfect matches can form
low stringency
below Tm- under conditions that stabilize double helix- imperfect bp can form
properties of chromosomal DNA
- each chromosome is single long polymer of DNA
- can be linear or circular
- GC content varies in different organisms (humans~40%) and varies non-randomly along human chromosomes (telomeres GC rich, centromeres AT rich)
- highly condensed DNA
- degree of condensation varies during cell cycle and along length of chromosome
topological stresss
- supercoiling
- positive is overwound, negative is underwound
- cut by topoisomerases during replication or transcription to relieve stress
- type I cuts 1 strand type II cuts both strands
structural features of RNA
- single stranded
- shorter than DNA
- complex tertiary structure
- unstable-vulnerable to base-catalyzed hydrolysis at 2’ hydroxyl
- pH above 7 RNA is degraded
- can for intramolecular H bonded bp-hairpin and stem loop
- secondary structure allows for well-defined shape that can be important for function and recognition-tRNA and ribozymes
mRNA
- 5% of total RNA, most heterogeneous in size of RNA types
- contains genetic info copied from specific regions of DNA to be used as a template for protein synthesis
ncRNA
- non-coding
- included miRNA, ribozymes
rRNA
- 80% of total RNA
- several species of distinct sized that are part of the structure of the ribosome
tRNA
- 15% of total RNA
- small RNA - 73-93 nucleotides
- contain elaborate secondary structures and some unique nucleotides
- serve as adaptor molecules in protein synthesis-recognize the code in mRNA indicating which aa comes next in a protein and bring that aa to the site of protein synthesis on ribosome
- have at least on specific tRNA molecule for each of the 20 aa
miRNA
- small endogenous RNA of 22 nt that play important regulatory roles in animal development
- bind to complementary sites of specific mRNA to inhibit their translation
siRNA
- small interfering
- 20-25bp dsRNA
- function in RNA interference 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
