Nucleic Acids and Proteins Flashcards
Describe the structure of DNA including the anti parallel strands, 5’-3’ linkages and hydrogen binding between purines and pyrimidines
- double strands run anti-parallel
- 5’ end has phosphate group attached to carbon 5
- 3’ end has hydroxyl group attached to carbon 3
- adenine and guanine are purines
- thymine and cytosine are pyrimidines
- 2 H-bonds between A and T
- 3 H-bonds between G and C
Outline the structure of nucleosomes
- core has 8 histones (4 types x 2)
- DNA wraps around core
- 5th type of histone holds nucleosome together
- DNA linkers connect DNA strand between nucleosome beads
State that nucleosomes help to supercoil chromosomes and help to regulate transcription
Nucleosomes help to supercoil chromosomes and regulate transcription.
Distinguish between unique or single-copy genes and highly repetitive sequences in nuclear DNA
Unique/Single Copy (genes)
- heritable factors that control a characteristic (coding)
- specific sequences of nucleotides within DNA
- contain exons/introns
Highly Repetitive (satellite)
- most of DNA in eukaryotes (5-45% of genome)
- not translated (non-coding)
- 5-300 base pairs long, duplicated as many as 10^5 times
State that eukaryotic genes can contain exons and introns
Eurkaryotic genes can contain exons and introns.
Explain DNA replication in terms of unwinding the double helix and separation of the strands by helicase, followed by formation of the new complementary strands by DNA polymerase
- during S-phase (interphase) before mitosis
- helicase separates strands
- starts at replication origin to replication fork (forms replication bubble)
- DNA polymerase adds builds complementary strand in 5’ to 3’ direction
- strand ending with 3’-hydroxyl uses continuous synthesis
- strand ending with 5’-phosphate uses discontinuous synthesis
Explain the significance of complementary base pairing in the conversation of the base sequence of DNA
- semi-conservative replication means 2 daughter molecules will have one old and one new strand
- base sequence is complementary in new strand
- A-T and C-G always go together because of H-bonds holding them together
State that DNA replication is semi-conservative
DNA replication is semi-conservative.
State that DNA replication occurs in a 5’ –> 3’ direction
DNA replication occurs in a 5’ to 3’ direction.
Explain the process of DNA replication in eukaryotes, including the role of enzymes (helicase, DNA polymerase, RNA primase, and DNA ligase), Okazaki fragments and deoxynucleoside triphophates
- Helicase breaks H-bonds between complementary parent strands; 2 strands ending at replication fork
- Gyrase cuts DNA to relieve tension and prevent tangling
- SSBs (single stranded binding proteins) anneal exposed ends to prevent H-bonding to each other
- RNA primase lays primers for DNA polymerase III to use as starting point
- DNA polymerase III adds deoxyribonucleoside triphosphates (ie. dATP) to 3’ end of new strand, using template strand as guide; energy from breaking 2Pi from triphosphate
- Leading strand (3’) builds continuously toward replication fork
- Lagging strand (5’) builds discontinuously away from replication fork; short segments called Okazaki fragments between primer
- DNA polymerase I excises RNA primers, replacing them with deoxyribonucleotides
- DNA ligase joins Okazaki fragments with phosphodiester bonds
- As 2 new DNA strands are synthesized, they twist into helix
- DNA polymerase I and III are exonucleases; remove incorrectly paired nucleotides to prevent further replication of mistake
State that DNA replication is initiated at many points in eukaryotic chromosomes
DNA replication is initiated at many points in eukaryotic chromosomes.
Compare the structure of RNA and DNA
DNA (deoxyribonucleic acid)
- deoxyribose (no -OH on C2)
- double-stranded (double helix)
- adenine, thymine, cytosine, guanine
- inside nucleus
RNA (ribonucleic acid)
- ribose
- single-stranded
- adenine, uracil, cytosine, guanine
- in/out of nucleus
Similarities:
- nucleotides linked by phosphodiester bonds, forming sugar-phosphate backbone
Outline DNA transcription in terms of the formation of an RNA strand complementary to the DNA strand by RNA polymerase
- RNA polymerase split double helix into 2 strands
- mRNA is transcribed from template to have complementary sequence
- U instead of T
Describe the genetic code in terms of codons composed of triplets of bases
- sequence of DNA nucleotides determine order of amino acids in polypeptide
- arranged in triplet codons so 3 nucleotides code for one of 20 amino acids
- genetic code is degenerate; more than on possible codon per aa (except methionine)
- reduces possible impact of mutations
Explain the process of translation, leading to polypeptide formation
Check 7.4.4
Discuss the relationship between one gene and one polypeptide
Gene in DNA:
- codes for the sequence of amino acids
- through transcription (nucleus) by RNA polymerase
mRNA (nuclear pores):
- translation (cytoplasm; rER) by ribosomes
Polypeptide Chain:
- folding (cytoplasm)
- based on # and type of amino acids
- quaternary proteins may have different polypeptides coded in more than one gene
State that transcription is carried out in a 5’ -> 3’ direction
Transcription occurs in a 5’ to 3’ direction.
Distinguish between the sense and antisense strands of DNA
- sense strand (coding) has same base sequence as mRNA but U instead of T
- antisense strand (template) is transcribed
Explain the process of transcription in eukaryotes, including the role of the promoter region, RNA polymerase, nucleoside triphosphates and the terminator
- Initiation
- RNA polymerase binds to DNA at the promoter region (usually string of A and T)
- unwinds and splits double helix into 2 strands - Elongation
- RNA polymerase pairs free nucleoside triphosphates to the antisense strand
- builds mRNA in 5’ to 3’ direction
- 2 phosphates are removed + converted to RNA nucleotides - Termination
- at end of gene, terminator sequence signals RNA polymerase to stop
- mRNA released; undergoes post-transcriptional modification before leaving nucleus
State that eukaryotic RNA needs the removal of introns to form mature mRNA
Primary transcript needs to become mature mRNA before translation
- spliceosomes cut out introns + rejoin exons
- add cap + tail to slow degradation by enzymes in cytoplasm
Explain that each tRNA molecule is recognized by a tRNA-activating enzyme that binds a specific amino acid to the tRNA, using ATP for energy
- tRNA is a cloverleaf with 3 loops
- each aa has specific tRNA-activating enzyme that attaches aa to 3’ end of tRNA molecule (CCA sequence)
- ATP is needed to bind aa to acceptor site + form peptide bonds
- anticodon loop has triplets of bases, complementary to codons on mRNA
- degeneracy; some aa have more than one tRNA
Outline the structure of ribosomes, including protein and RNA composition, large and small subunits, three tRNA binding sites and mRNA binding sites
- small and large subunit, made of proteins and rRNA
- mRNA binding site is in small subunit
- A site (aminoacyl) holds tRNA carrying aa to be added to chain
- P site (peptidyl) holds tRNA carrying polypeptid chain
- E site (exit) where empty tRNA leaves ribosome
- only 2 tRNA are on ribosome at once
State that translation consists of initiation, elongation, translocation and termination
Translation consists of initiation, elongation, translocation and termination
State that translation occurs in a 5’ –> 3’ direction
Translation occurs from 5’ to 3’
Draw and label a diagram showing the structure of a peptide bond between two amino acids
-OH attached to C of 1st aa removed
-H attached to N of 2nd aa removed
H2O produced
Explain the process of translation including ribosomes, polysomes, start codons and stop codons
- Initiation
- initiator tRNA (methionine) with anticodon UAC binds to small subunit
- small subunit binds to 5’ of mRNA
- moves along mRNA until reaches start codon (AUG)
- large subunit binds
- initiator tRNA starts in P site - Elongation
- next tRNA with complementary anticodon binds to A site
- ribosome catalyzes peptide bond between aa in A and P sites
- aa in P site detaches from tRNA
- tRNA in P site moves to E site and leaves
- tRNA in A site is translocated to P site
- mRNA shifts by one codon
- peptide chain elongates as ribosome moves along mRNA (5’ to 3’) - Termination
- translocation/elongation continue until stop codon
- no tRNA has anticodon for stop codon
- protein called release factor attaches to stop codon in A site
- large subunit advances over small subunit
- polypeptide released
- last tRNA detaches and ribosome comes apart
State that free ribosomes synthesize proteins for use primarily within the cell and that bound ribosomes synthesize proteins primarily for secretion or for lysosomes
- free ribosomes synthesize proteins for use in cell
- membrane-bound ribosomes synthesize proteins for secretion
Outline the use of polymerase chain reaction (PCR) to copy and amplify minute quantities of DNA
- make billions of identical copies from small source (trace) DNA within few hours
- useful for DNA sequencing and profiling (require a lot of DNA)
- samples from crime scenes (eg. blood, semen) are too small to analyze
- also called DNA amplification; repeating cycles of PCR replicate DNA at 2^n where n = #cycles
State that, in gel electrophoresis, fragments of DNA move in an electric field and are separated according to their size
- DNA fragments pass through gel (agarose) that separates according to size
- DNA has -ve phosphate groups, electric field causes fragments to move from wells at -ve end to +ve end
- fragments are made by PCR or mixing with endonucleases
- molecular standard ladder creates scale so length can be estimated
- bands are stained to determine if DNA are related
State that gel electrophoresis of DNA is used in DNA profiling
- used to identify individuals according to variation in sequences of satellite DNA
- amplified by PCR and cut into fragments by restriction enzymes
- lengths vary between individuals
- pattern of bands from gel electrophoresis unlikely to be same for unrelated people
Describe the application of DNA profiling to determine paternity and also in forensic investigations
- Criminal investigations/forensics
- DNA isolated from cells collected at crime scene (eg. blood, skin, semen) and amplified using PCR
- run using gel electrophoresis
- profiles compared to samples from victims/suspects
- banding pattern can be matched - Paternity suits
- DNA collected from mother, child, suspected father
- banding pattern of child will have both parents - Other
- identification of people who died long ago
- determine if twins are identical
- determine variation in DNA between different populations
- track migration patterns of groups of people over time
Analyse DNA profiles to draw conclusions about paternity or forensic investigation
- # human genes
- location of specific genes
- discovery of proteins + functions
- evolutionary relationships
Outline three outcomes of the sequencing of the complete human genome
- study of how genes influence human development by comparing sequences between individuals (ie. autism, height, hair, eye, skin)
- identification of genetic diseases (eg. cystic fibrosis, cancer, heart disease, Huntington’s)
- new insights into origins, evolution, migrations of humans using mitochondrial DNA (mtDNA passes mostly from mother)
- production of new drugs
- comparison to DNA sequences of other species (eg. cytochrome c oxidase); DNA barcoding
State that, when genes are transferred between species, the amino acid sequence of polypeptides translated from them is unchanged because the genetic code is universal
- recombinant DNA is fragment of DNA spliced into another species
- aa sequence is translated from gene of one to another because code is universal
Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast or other cell), restriction enzymes (endonucleases) and DNA ligase
- Restriction Enzymes (endonucleases)
- cleave double-stranded DNa into fragments at reaction sites
- isolated from and named according to bacteria where they’re found
- have recognition sites that are 4-8 base pairs long and palindromic
- bind to recognition sites on DNA to break phophodiester bonds by hydrolysis
- create blunt ends when cut in same spot on both strands (DNA remain fully paired)
- create sticky ends where short strands of unpaired bases (disrupt H-bonds); more successful for recombinant DNA - Plasmids
- small circular pieces of DNA, in addition to main circular chromosome in bacteria (eg. E.coli)
- isolated to provide way to transfer foreign genes
- restriction enzyme opens plasmid at target sequence; foreign gene spliced into plasmid at sticky ends where complementary bases form H-bonds
- DNA ligase forms phosphodiester bonds in sugar-phosphate backbone
- forms recombinant plasmid containing foreign gene
- placed in solution with host cells which undergo transformation (takes in plasmid)
- replicate when bacteria divide so all offspring express foreign gene
- used to splice human insulin gene into E.coli
State two examples of the current uses of genetically modified crops or animals
- deactivation of ripening gene in tomatoes; delay ripening and extend shelf-life
- splicing gene from Bt into corn; resistant to corn borers
- transgenic sheep enhance wool by increaseing amount of cysteine made
- modify fatty acid content of soybeans to increase oleic acid (reduce need for hydrogenation) and decrease linolenic acid (improve flavour stability)
- lectin transferred from pea plants to potatoes; disrupts digestive system so leaves protected from insects
Discuss the potential health benefits and possible harmful effects of one example of genetic modification
- Bt (Bacillus thuringiensis) gene transferred to maize, kills corn borers
Pros:
1. Less pest damage so higher crop yields (reduce food shortage)
2. Less land needed for crops so more land for wildlife conservation
3. Less use of insecticide sprays which are expensive and harmful to humans/wildlife
Cons:
- Humans/animals who each modified maize can be harmed by bacterial DNA/Bt toxin
- Non-pest insects can be killed if pollen with toxin blows onto wild plants (ie. Monarch butterflies can die even though do not feed on maize)
- Pop. of wild plants might change; cross-pollination may affect some but not others so wild plants with Bt gene will have advantage over others
Define clone
Clone - a group of genetically identical organisms or a group of cells artificially derived from a single parent cell
Outline a technique for cloning using differentiated animal cells
?
Discuss the ethical issues of therapeutic cloning in humans
For:
- DNA/proteins are unlikely to cause problems because digested in human gut
- GMOs do not survive long in natural ecosystem; natural selection acts against them
- Increase yield and reduce production cost (increase food supply/farmers profits, reduce food cost)
- Transfer of genes is natural phenomenon
Against:
- Uncertain about long-term effects on human health from genes in GMOs
- Genes could escape and transfer to wild organisms, creating “superweeds” or damaging ecosystems
- May only develop to suit conditions on large commercial farms, harder for small farms to compete
- Moving genes between species infringes right of independent existence
