Week 12 Flashcards
1
Q
What major breakthroughs in the 19th Century, gave clue that DNA is our inherited genetic material?
A
Mendel (1822-1884): Discovery of dominant and recessive traits.
Darwin (1809-1822): Natural selection theory of evolution
Miescher (1844-1895): Discovery of nucleic acid
2
Q
Who discovered nucleic acid?
A
Friedrich Miescher (1844-1895).
Originally trained as a doctor but decided he wanted to do research instead, and was interested in the biochemistry of cells; particularly nuclei
3
Q
How did Miescher discover nucleic acid?
A
Took pus from used bandages, used the wbc to find purified nuclei.
Extracted the nuclei and found a precipitate rich in phosphorus (P) and nitrogen (N) - “nuclein”
N-rich fraction = protein
Novel substance: acidic P-rich fraction = “nucleic acid”
4
Q
Miescher and other scientists discovered nucleic acids consists of repeating units of what?
As well as two types of nucleic acid, which was what?
A
- Five carbon sugar
- Nitrogenous bases
- Phosphate
- RNA (in nucleoplasm, not chromosomes)
- DNA (in chromosomes)
5
Q
Experiment 1: what is genetic material? What did Griffith’s transformation experiment look at?
A
- Used two strains of S. pneumoniae, (R) that’s benign and (S) that’s virulent
- Injected different forms into mice. (S, R, Killed-S, Killed-S + R)
6
Q
Experiment 1: what is genetic material? What did Griffith’s transformation experiment find?
A
Mice died due to their blood containing live pathogenic strains of S. pneumoniae, even tough a Killed-S form and R- form were injected.
7
Q
Experiment 1: what is genetic material? What did Griffith’s transformation experiment conclude?
A
There must be a ‘transforming principle’, as the ‘genetic material’ was capable of reprogramming R-form cells into S-form cells which were disease causing.
But, he didn’t know what caused this transformation.
8
Q
Experiment 2: what is genetic material? Oswald Avery (1944) studied?
A
Built on Griffiths by subjecting the heat-killed S-type bacteria to a range of tests to identify the nature of the ‘transforming principle’
9
Q
Experiment 2: what is genetic material? Oswald Avery (1944) found?
A
The transforming principle was:
* Resistant to proteases, lipases and ribonucleases, so not protein, lipid or RNA
* Ethanol-insoluble, so not carbohydrate
* It had a high molecular weight like DNA
* Had a positive reaction to the Dische test for deoxyribose of DNA
10
Q
Experiment 2: what is genetic material? Oswald Avery (1944) conclude?
A
The transforming principle was DNA.
But, some people were sceptical so further experiments were conducted by Hershey-Chase!
11
Q
Experiment 3: what is genetic material? Hershey-Chase (1952) studied what?
A
Used bacteriophages under an electron microscope to show that the virus doesn’t enter the cell. Rather it binds outside and injects its ‘genetic material’ and later the cell is reprogrammed to produce viral particles
12
Q
Experiment 3: what is genetic material? Hershey-Chase (1952) results?
A
- Phage protein labelled with 35S - Most of the radioactivity in supernatant (Containing phage ghosts)
- Phage DNA labelled with 32P - Most of the radioactivity in the pellet (containing the intact cells)
13
Q
Experiment 3: what is genetic material? Hershey-Chase (1952) concluded?
A
DNA is injected into the cell, not protein
14
Q
What was did Chargaff do (1950)?
A
He reasoned that the 4 nitrogenous bases (adenine, guanine, thymine, cytosine) must contain genetic code as the sugar and phosphate groups were invariant.
He examined ratios of these in DNA of various organisms
15
Q
What did Chargaff find (1950)?
A
- Ratio of the four bases is no 1:1:1:1 (suggests it is not simply a structural molecule)
- Ratio is species-specific
- Base composition always obeys a strict rule: A=T and G=C
16
Q
Who solved the structure of DNA (1953)?
A
Watson & Crick
Who used Franklins x-ray diffraction photograph of DNA structure (1952). (Given by Wilkins without her knowing)
17
Q
What are the three components of a DNA nucleotide?
A
- Pentose sugar
- Bases (A, T, G, C)
- Phosphate
(5’ located on the CH2, 3’ located on the HO on the pentose)
18
Q
What DNA bases are purines?
A
Adenine, Guanine
19
Q
What bases are pyrimidines?
A
Cytosine, Thymine
20
Q
What bond joins a polynucleotide chain (DNA sugar-phosphate backbone)
A
Phosphodiester bond
21
Q
What did Watson and Crick conclude from the x-ray diffraction of DNA?
A
- It is a helix
- 2nm wide (with 2 nucleotide chains)
- Length of each turn is 3.4nm
- Distance between repeating units is 0.34nm
- Therefore 10 nucleotide pairs per turn
- Purines must be paired with pyrimidines
- The two strands are anti-parallel
22
Q
What bond ensures specific base pairing?
A
Hydrogen bonds
(3 for G-C base pair (stronger))
(2 for A-T base pair (weaker))
23
Q
What is the transforming principle?
A
DNA
24
Q
What is the distance between base pairs in the DNA double helix?
A
0.34nm
25
The haploid human genome contains ~3 billion base pairs. What length of dsDNA is there in 1 diploid cell?
2m
26
Watson & Crick suggested that each strand of DNA can act as a template for the synthesis of a new complementary strand. What did they call this?
Semi-conservative replication!
27
Semi-conservative replication: Experimental proof study?
Meselson-Stahl experiment
1. Bacteria cultures in medium with 15N (heavy isotope)
2. Bacteria transferred to medium with 14N (lighter isotope)
3. DNA sample centrifuged after first replication
Found all DNA of intermediate density = 1 light strand and 1 heavy strand.
28
What does DNA polymerase require?
- Single-stranded template DNA
- All four nucleoside triphosphates (dNTPs)
- Free 3' hydroxyl (primer)
29
What is the role of DNA polymerase in semi-conservative replication?
- Synthesises DNA in 5' to 3' direction
- Inserts complementary nucleotides
- Uses energy from breaking phosphate bonds
- "Proof-reading" ability - can remove incorrectly inserted nucleotides
30
Where does semi-conservative replication begin?
At the origin of replication, which forms a bubble as a daughter strand begins to form on the template before straightening into two daughter DNA molecules.
31
Each replication fork has 2 types of strands, what are these known as?
Leading and lagging
32
Synthesis of the leading strand: step one (semi-conservative replication)?
Helicase unwinds and separates the two strands.
Single stranded binding proteins prevent DNA strands from re-annealing
33
Synthesis of the leading strand: step two (semi-conservative replication)?
RNA primer synthesised and added by 'primase'.
DNA polymerase III needs a 3' OH group
34
Synthesis of the leading strand: step three (semi-conservative replication)?
Leading strand synthesised continuously in 5' to 3' direction
35
Synthesis of the lagging strand: step one (semi-conservative replication)?
1. Primase joins RNA nucleotides into a primer
2. DNA pol III adds DNA nucleotides to the primer, forming Okazaki fragment 1.
36
Synthesis of the lagging strand: step two (semi-conservative replication)?
After reaching the next RNA primer to the right, DNA pol III detaches.
Fragment 2 is primed, then DNA pol III adds DNA nucleotides, detaching when it reaches the fragment 1 primer.
37
Synthesis of the lagging strand: step three (semi-conservative replication)?
DNA pol I replaces the RNA with DNA, adding nucleotides to the 3' end f the fragment 1 (and later, of fragment 2)
38
Synthesis of the lagging strand: step four (semi-conservative replication)?
DNA ligase forms a bond between the newest DNA and the DNA o fragment 1.
The lagging strand in this region is now complete.
39
What is the role of helicase in DNA replication?
Unwinds the helix to open it up
40
What is the role of single-strand binding proteins in DNA replication?
Hold the helix open
41
What is the role of primase in DNA replication?
Synthesises the RNA primers needed for initiation of DNA synthesis
42
What is the role of DNA Polymerase III in DNA replication (in prokaryotes; similar proteins do the same job in eukaryotes)?
Extends the DNA (or RNA) strand from the 3' end, copying the template
43
What is the role of DNA polymerase I in DNA replication (in prokaryotes; similar proteins do the same job in eukaryotes)?
Removes the RNA primer and fills in gaps between Okazaki fragments
44
What is the role of DNA ligase in DNA replication?
Seals the gaps between Okazaki fragments
45
Who was the first to connect inherited human disorders with Mendel's laws of inheritance?
Archibold Garrod who was interested in diseases where the patient is unable to carry out a particular biochemical reaction so studied Inborn Errors of Metabolism (1902)
46
What disease did Garrod study? What did he want to understand?
Alkaptonuria
- Urine darkens from yellow to black when exposed to air
- Urine from these patients contains large amounts of homogentisic acid (originally called alkapton)
He wanted to know how this disease came about as it ran in families!
46
Archibald Garrod found what about the disease studied?
By using punnet squares he was able to note it was an inherited disease; inherited in an autosomal recessive fashion
47
What did Garrod hypothesize?
Alkaponuria patients lack the enzyme necessary for breaking down homogentisic acid
Lack of the enzyme is due to a defect in a gene (now we know this missing enzyme is homogentisic acid oxidase)
48
What did Garrod conclude from his Alkaptonuria experiment?
Defects in genetic material can lead to specific diseases which can be inherited
Mendelian Genetic inheritance can be observed in humans
Lack of an enzyme is due to a defect in a gene
49
What was Beadle and Tatum's "one-gene, one-enzyme" experiment?
Identified new recessive mutations require at least two generations of crosses as most higher organisms have diploid genomes.
Used a simple organism: Neurospora (bread mold), finding that after the haploid stage of life cycle recessive traits will appear in offspring.
50
What did Beadle and Tatum theorize?
Normal Neurospora can grow without many nutrients because it has enzymes to generate its won, so they theorized that if they damaged the DNA for one of these enzymes then the cells wouldn't be able to grow without the particular nutrient.
51
What was Beadle and Tatum's test for their experiment?
To test if genes were responsible for specific chemical reactions they irradicated Neurospora with X-rays
Identifying a series of auxotrophic mutants. (mutant strains that cannot synthesize a particular molecule required for growth, therefore they will only grow if they are supplied with that molecule)
52
How can auxotrophic mutants be screened for (Beadle and Tatum)?
1. Culture individual spores on complete medium
2. Transfer to minimal medium (MM) to identify possible auxotrophs
3. Test candidates for growth on MM supplemented with different classes of nutrients
4. Test candidates for growth on MM supplemented with individual amino acids
5. Identify the amino acid that allows your mutant to grow
53
What did Srb and Horowitz do?
Took Beadle and Tatum's experiment slightly further and identified three classes of arginine auxotroph by testing metabolic pathways.
54
What did Beadle and Tatum propose?
That each enzyme in a pathway was controlled by one gene.
Role of a gene is to encode an enzyme - and that for each enzyme there is a gene
"One Gene - One Enzyme" Hypothesis
55
What has occurred since the One Gene - One Enzyme hypothesis?
Noted that not all gene products are enzymes. Therefore it must be "One Gene - One Protein"
But further work showed some proteins are made of more than one polypeptide chain, therefore it must be "One Gene - One Polypeptide"
56
How do genes specify proteins?
DNA in nucleus but proteins made in cytoplasm
Genes do not specify proteins directly
"Messenger" required - information transferred from nucleus to cytoplasm
Messenger is ribose nucleic acid (RNA)
57
RNA structure?
A polymer of nucleotides containing ribose sugar and combinations of four bases: adenine, guanine, cytosine and uracil - usually single-stranded
58
What experiment provided evidence for mRNA?
Pulse-chase experiments, as they show RNA is made in the nucleus and moves into the cytoplasm
59
What are the two main steps in the process of gene expression?
Transcription - DNA to mRNA
Translation - mRNA to Protein
60
What is the process of DNA to mRNA to protein known as?
Central Dogma
61
What is transcription?
The synthesis of mRNA molecule using one strand of DNA as a template such that G pairs with C and A pairs with U
Transcription is catalysed by RNA Polymerase
62
What are the three stages of transcription?
Initiation
Elongation
Termination
63
Stage one of Transcription: initiation?
RNA polymerase binds to the promoter region of the DNA and unwinds the promoter region. Finding the RNA transcript on the template strand.
64
Stage two of Transcription: elongation?
RNA polymerase moves along the DNA template, unwinding the double helix and catalysing the addition of ribonucleotides to the 3' end of the growing RNA molecule
65
Stage three of Transcription: termination?
RNA polymerase reaches a termination signal in the newly produced RNA (terminator). DNA is rewound and a complete RNA transcript has been formed.
66
How may different amino acids can be encoded from a strand of mRNA?
20, using a 3-letter coded strand of mRNA during translation
67
What did Francic Crick and Sydney Brenner prove?
That 'codons' are three letters
68
How did Crick and Brenner prove that 'codons' are three letters?
The first codon proved was UUU, coding for phenylalanine. They took artificial mRNA consisting entirely of uracil and added to a cell-free translation system, finding the protein consisted entirely of phenylalanines.
More experiments had to be performed to work out the remainder of the codons.
69
What three codons specify 'stop codons' to signal the end of translation?
UAA, UAG, UGA
69
What codon has the dual role of the start codon and also encodes methionine?
AUG
70
How is genetic code specific but redundant?
Each codon can only specify one amino acid so it is specific,
But amino acids can be specified by more than one codon so it is redundant
71
Why is genetic code (almost) universal?
The same codons are used for the same amino acids from bacteria to humans. Suggesting all life has a common evolutionary ancestor.
(A few rare exceptions include: mitochondrial genomes, some stop codons)
72
What adapters are required to link mRNA and amino acids? Why?
Transfer RNAs (tRNAs) because mRNA cannot act as a physical template for amino acids
73
What is the structure of tRNA?
~80 nucleotides in length
Single-stranded but base pairs form within the chain
Clover leaf structure further folds to make L-shaped molecule
Anticodon is at one end - base-pairs with codon
Amino acid attachment site is 3' hydroxyl group at end of RNA chain
74
Why is tRNA specific?
Each tRNA is specific for a single amino acid determined by its anticodon as tRNAs are the adaptors
75
How does a tRNA become attached to the correct amino acid?
Specific attachment is carried out by amino-acyl tRNA synthetases (activating enymes)
76
How many codons are there in genetic code compared to tRNAs?
61 amino acid codons and only 40-45 tRNAs
77
What is the ribosome composed of?
ribosomal RNA (rRNA) and proteins; it has two subunits - large and small
78
What does ribosomes bind?
Binds mRNA and amino acyl-tRNAs
79
What are the three tRNA binding sites called?
What do they catalyse?
Three tRNA binding sites:
A (Aminoacyl-tRNA binding site)
P (Peptidyl-tRNA binding site)
E (Exit site)
Catalyses stepwise formation of peptide bonds (amino acids added from N terminus to C terminus)
80
What direction does the ribosome move along the mRNA?
5'-3' direction.
By recognising the correct start codon, ribosomes ensure correct reading frame is used
81
What three stages does protein synthesis occur in? Summarise these stages?
Initiation - small subunit binds mRNA and initiator amino acyl-tRNA then large subunit binds
Elongation - peptide bonds are formed as ribosome moves along the mRNA
Termination - one of the three stop codons enters A-site and the completed protein is released
82
Step one of protein synthesis: Initiation?
1. Small ribosomal subunit binds mRNA near its 5' end
2. Initiator tRNA bind to AUG start codon
3. Large subunit binds so that the initiator tRNA fits into the P-site on the large subunit
83
What does initiation of protein synthesis require?
This requires energy from GTP hydrolysis and proteins called initiation factors (which help stabilise initiator tRNA and to assemble ribosome)
84
Step two of protein synthesis: Elongation (1)?
1. Incoming amino-acyl tRNA base pairs with codon in the A-site (Requires hydrolysis of GTP)
2. Peptide bond formed between amino group of new amino acid and the COOH group of amino acid in the P-site (catalysed by peptidyl transferase)
85
Step two of protein synthesis: Elongation (2)?
3. Growing polypeptide chain now in the A-site
4. Translocation - tRNA in the P-site is ejected and the ribosome moves along the mRNA by precisely one codon (requires hydrolysis of GTP)
86
Step two of protein synthesis: Elongation (3)?
Growing chain now in the P-site and the A-site is free to accept the next incoming aminoacyl tRNA
87
Three steps of the elongation cycle in protein synthesis?
Codon recognition,
Peptide bond formation,
Translocation
88
What is the peptide bond formation in the elongation cycle of protein synthesis?
Catalysed by peptidyl transferase - an RNA enzyme (ribozyme)
Proteins grow from amino (N) terminal to carboxy (C) terminal
89
Step three of protein synthesis: Termination?
- Stop codon in A-site
- There are no tRNAs for stop codons
- Water added to end of polypeptide chain
- Completed polypeptide released from tRNA in P-site
- Ribosome dissociates, 2X GTP hydrolysed
90
What causes 'wobble' base-pairing
Flexibility within a codon, the third base, so it can be one of two eg, U or C when coding occurs.
91
Protein synthesis in prokaryotes versus eukarytes?
Eukaryotes = Nuclear membrane, several organelles involved (proteins are trafficked to correct site)
Prokaryotes = No nuclear membrane so transcription and translation are coupled, no organelles (proteins diffuse through cytoplasm)
92
What is coupled transcription and translation (prokaryotes only)?
Several RNA polymerase transcripting a single strand of DNA with polyribosomes attached translating mRNA at the same time.
93
What are the two types of DNA mutations?
Point mutation - Change in nucleotide sequence
Chromosomal mutation - Change in gene position/number
94
Mutations in somatic tissues?
- Not passed onto offspring
- Passed onto all cells descending from original mutant
- ~85% cancers caused by this mutation
95
Mutations in germ line tissues?
- Passed on to offspring
- Cause of inherited genetic diseases
- Raw material from which natural selection produces evolutionary change
96
Define mutation
A permanent change in the DNA of a cell
97
What are binding sites on tRNA used for?
Specific tRNA(s)
Corresponding amino acid (one for each of the 20 amino acids)
ATP
98
What are the 2-step attachment processes when attaching an amino acid to its tRNA?
ATP hydrolysed and amino acid joined to AMP
Correct tRNA binds and amino acid transferred from AMP to the tRNA
99
How are certain proteins targeted for secretion to the endoplasmic reticulum (in eukaryotes only) step one?
Polypeptide synthesis begins on a free ribosome in the cytosol
An SRP binds to signal peptide, halting synthesis momentarily
100
How are certain proteins targeted for secretion to the endoplasmic reticulum (in eukaryotes only) step two?
The SRP binds to a receptor protein in the ER membrane. This receptor is part of a protein complex that has a membrane pore and a signal-cleaving enzyme
101
How are certain proteins targeted for secretion to the endoplasmic reticulum (in eukaryotes only) step three?
The SRP leaves, and polypeptide resumes growing, meanwhile translocating across the membrane. (The signal peptide stays attached to the membrane)
102
How are certain proteins targeted for secretion to the endoplasmic reticulum (in eukaryotes only) step four?
The signal-cleaving enzyme cuts off the signal peptide
The rest of the completed polypeptide leaves the ribosome and folds into its final conformation
103
What are the two types of point mutations?
Base pair substitutions,
Base pair insertion/deletion (Usually very damaging)
104
Mutations can be spontaneous or induced. What is meant by this?
Spontaneous: through DNA replication (can also occur due to inherent instabilities in DNA)
Induced: by mutagens such as chemicals (base analogues, modifying agents, intercalating agents) or physically (ionising radiation, ultraviolet radiation)
105
How do spontaneous mutations occur?
Nucleotides can change to other conformations eg isomers and tautomers
During DNA replication an incorrect base is inserted to form mismatched pair
106
Induced DNA mutations: What are the three types of chemical agents that act on DNA resulting in mutations?
- Chemicals that resemble DNA bases but pair incorrectly when incorporated in DNA (base analogues)
- Chemicals that remove the amino group from adenine or cytosine eg nitrous acid / nitrite
- Chemicals that add hydrocarbon groups to nucleotide bases
107
What are intercalating agents? What do they cause?
Insert between bases and distort DNA helix,
Interfere with replication.
They tend to cause frameshift mutations
108
How does ionising radiation cause mutations to DNA?
1. Directly ionises DNA
2. Ionises water to produce free radicals
It can damage bases and cause double-stranded breaks!
109
How does ultraviolet radiation cause mutations within DNA?
- Absorbed specifically by the pyrimidine bases (cytosine and thymine)
- Covalent bonds can form between adjacent T or C nucleotides forming pyrimidine dimers
- Blocking DNA synthesis, leaving a gap opposite the site of damage
110
How can Thymine dimers be removed by nucleotide excision repair (NER)?
- Broad specificity repair system recognises distortions in the DNA helix
- Individuals with the rare hereditary disorder called xeroderma pigmentosum are deficient in NER.
- XP characterised by development of skin cancer at an early age but only those parts of body exposed to sun
111
Mutations in which two genes result in the formation of cancer?
oncogenes,
tumour suppressors
112
Most cancers occur due to mutations in which type of cells?
Somatic cells. They are not inherited
113
What % of cancers are caused by inherited mutations? Why?
~15% as the inherited mutations give a pre-disposition to cancer (known as cancer predisposition syndromes) such as xeroderma pigmentosum
114
What is the Ames test used for?
To identify mutagenic properties within an organism by using liver extract as liver extract contains enzymes that may convert non-mutagens into mutagens
