Nucleic Acids and Gene Expression Flashcards
1
Q
What is a nucleotide made up of?
A
Nitrogenous base + Sugar (Deoxyribose) + Phosphate
2
Q
What sugar is present in RNA?
A
Ribose
3
Q
What bases are purines?
A
Adenine and Guanine
4
Q
What bases are pyrimidines?
A
Cytosine, Thymine and Uracil
5
Q
What bases makeup RNA
A
A, C, U and G
6
Q
What is a nucleoside made up of?
A
Nirogenous base + Sugar
7
Q
What does DNA helicase do?
A
Uses ATP to provide energy to break hydrogen bonds to unzip DNA
8
Q
What does DNA polymerase do?
A
Synthesises a new strand of DNA. It cannot initiate replication
9
Q
Which direction is DNA synthesised in?
A
5’ to 3’ direction
10
Q
What is the leading strand?
A
DNA strand that is continuously synthesised
11
Q
What is the lagging strand?
A
Strand of DNA that is synthesised in Okazaki fragments
12
Q
What does DNA ligase do?
A
Joins Okazaki fragments to the previous one
13
Q
What does DNA primase do?
A
Synthesises a short fragment to initiate DNA replication
14
Q
What enzyme proof-reads the DNA and removes any incorrect bases?
A
DNA polymerase
15
Q
Where does DNA replication start in E.coli?
A
Ori.C
16
Q
How long is M phase
A
1 hour
17
Q
How long is G1 phase?
A
10 hours
18
Q
How long is S phase and what occurs here?
A
9 hours, DNA synthesis
19
Q
How long is G2 phase?
A
4 hours
20
Q
What is G1 phase prior to?
A
S phase (DNA synthesis)
21
Q
What is G2 phase between?
A
Synthesis and mitosis
22
Q
What cells are in G0 phase
A
Cells that no longer divide
23
Q
Are the chromosomes visible in interphase?
A
No
24
Q
What occurs in prophase?
A
Chromosomes condense and each contain two sister chromatids
25
What occurs in metaphase?
Condensed chromosomes align on central plane of spindle of microtubules
26
What occurs in anaphase?
Sister chromatids separate and are pulled to spindle poles
27
What occurs in telophase?
Sister chromatids move to opposite poles of spindle
28
What occurs in cytokinesis?
Division of cytoplasm
29
What occurs in G1 interphase?
The condensation process is reversed
30
What is the difference between DNA and RNA
RNA has ribose instead of deoxy ribose
| RNA has uracil instead of thymine and is usually single standed
31
What is synthesised by RNA polymerase I?
rRNA
32
What is synthesised by RNA polymerase II?
mRNA
33
What is synthesised by RNA polymerase III?
tRNA
34
What is the sequence in DNA that brings about transcription?
Promoter regions
35
Is mRNA produced the same as the sense or antisense strand?
Sense
36
Ribonucleotide bases are joined together with what bond?
Phosphodiester bonds
37
What transcription factor activates gene expression?
Transcription activators
38
What is transcription repressors and what does it do?
Transcription factor that suppresses gene expression
39
What is the first protein to bind in the basal transcription complex?
Transcription complex II D (TFIID)
40
Which transcription factor binds to TFIID and RNA polymerase II
TFIIB
41
What does NFκB transcribe?
Cytokines for inflammation
42
How does aspirin stop inflammation?
Inhibits the breakdown of IκB so NFκB remains in the cytoplasm and is unable to initiate transcription of cytokines
43
How many types of RNA polymerase do bacteria have?
One
44
What is heterogenous nuclear RNA?
pre-mRNA
45
Are introns or exons used to transcribed the final RNA?
Exons
46
What sequence do introns start and end with?
Start with GU
| End with AG
47
What is the splice donor sequence?
AGGU
48
Explain the process of removal of introns?
1) U1 binds to the splice donor sequence (AGGU)
2) U2, U4 and U6 bind to the intron
3) U5 binds to the splice acceptor site Pyr15NCAG
This completes formation of the spliceosome and causes cleavage of the sequence at the end of the exon and beginning of the intron
4) Free end of intron folds back to specific A in the intron known as the branchpoint
5) The branch results from a phosphodiester bond between 5' phosphate on G and the 2' OH of A creating a loop
6) Phosphodiesterase bond between G at the end of the intron and next exon is cleaved and the intron is removed as "lariat" structure
7) The exposed, adjacent exon sequenced are finally ligated together
49
What is the first post-transcriptional modification of RNA?
Addition of a 5' cap
50
Explain the process of addition of a 5' cap?
1) Hydrolysis of the triphosphate on the 5' end of the mRNA to form a diphosphate (by a phosphatase enzyme)
2) The diphosphate reacts with the α-phosphate of GTP, to form a 5'-5' phosphate linkage (by guanylyltransferase)
3) The cap if further modified at N7 position in the purine ring to form 7-methyl guanylate cap (by methyl transferase)
Cap protects mRNA at 5' end and greatly enhances translation
51
What enzyme causes hydrolysis of the triphosphate on the 5' end of the mRNA?
A phosphatase enzyme
52
What enzyme forms a 5'-5' phosphate linkage by reacting the diphosphate with the α-phosphate of GTP?
Guanylyltransferase
53
What enzyme modifies the 5' cap at the N7 position in the purine ring to form 7-methyl guanylate cap?
Methyl tranferase
54
How does polio take over the nervous system and cause paralysis in hours?
Makes an enzyme that cleaves the 5' cap and replaces it with its own so it is no longer a template for translation
55
What enzyme ligates together adjacent exons?
Ligase
56
What is the splice donor sequence?
AGGU
| Ends the exon and starts the intron
57
What is the splice acceptor sequence?
Pyr15NCAG
| Ends the intron
58
What is the process of adding the poly-A tail?
1) RNA polymerase reaches the end of the gene it is transcribing and recognises a sequence in the genome
2) RNA binding proteins that travel with RNA polymerase recognise this, in particular, Cleavage Stimulating Factor (CstF) and Cleavage and Polyadenylation Specificity Factor (CPSF).
3) Once the bind to a specific sequence, additional proteins assemble to create the 3' end of the mRNA.
4) Poly-A polymerase (PAP) adds (one at a time) 200 A nucleotides to the end of the mRNA sequence
59
What is CstF?
Cleavage Stimulating Factor
60
What is CPSF?
Cleavage and Polyadenylation Specificity Factor
61
What is PAP?
Poly-A Polymerase
62
What causes β-thalassaemia?
A relative deficiency of β-chains.
Several types of β-thalassaemia feature splice site mutations in the β-globin gene
The intron cannot be spliced so mature mRNA is not formed and translation of a non-functional occurs
63
What is Thalassaemia?
A genetic, haematological disease in which there is an imbalance in the relative amounts of α-chains and β-chains making up haemoglobin
64
What does microRNA do?
Control the translation of most genes
65
What does siRNA/RNAi do?
Viral defence, experimental tool
66
What does piRNA do?
Clearly important for germ cell production
67
What does long ncRNA do?
Xist- Important for X chromosome inactivation
68
What is lyonisation?
The process where one of the female X chromosomes is inactivated. It is packaged so it has transcriptionally inactive heterochromatin. The choice of which X chromosome is inactivated is random.
69
What is a codon?
A group of three nucleotides
70
Protein synthesis always starts with what?
Met = AUG
71
What are the stop codons?
UAA, UAG, UGA
72
What is the typical structure of mRNA?
5' cap - 5' UTR - Coding Region - 3' UTR - poly A
73
What is the cap in mRNA?
7-methyl guanosine
74
What is "wobble" with regard to translation?
The first two bases in a codon are really important with respect to binding energy of mRNA. The wobble is the third base which has a lower binding energy so may accept a different base, resulting in a different primary sequence
75
What is stringency?
The conditions in a reaction (temperature, salt and pH) that dictate the annealing of single-stranded DNA.
High stringency only allows duplexes to form between strands with perfect complementarity
Lower stringency allows annealing between strands with some degree of mismatching
76
What is a hybridisation probe?
A fragment of DNA or RNA of variable length (usually 100-1000) bases long) which is radioactively labelled. It can then be used in DNA or RNA samples to detect the presence of nucleotide sequences.
The probe hybridises to single-stranded nucleic acid (DNA or RNA) whose base sequence allows probe-target base pairing due to complementarity between probe and target
77
Explain the process of hybridisation and blotting?
The labelled DNA or RNA probe is first denatured (by heating or alkaline conditions such as sodium hydroxide) into single stranded DNA (ssDNA) and then hybridised to the target ssDNA (Southern Blotting) of RNA (Northern Blotting) immobilised on a membrane or in situ
78
The energy needed to denature the probe DNA is performed to disrupt the hydrogen bonds between the bases. What does this depend on?
Strand length
Base composition
Chemical environment
79
Why does probe DNA strand length affect the energy require to denature it?
A longer strand has more hydrogen bonds to break
80
Why does probe DNA base composition affect the energy required to denature it?
G-C pair has one more hydrogen bond than A-T so it is harder to break
81
How many hydrogen bonds to A-T make?
2
82
How many hydrogen bonds do G-C make?
3
83
Why does the chemical environment affect denaturing of a chemical probe?
Monovalent cations (Na+) stabilise the DNA duplex by neutralising the charge on phosphate backbone. Denaturants (formaldehyde/urea) destabilise the DNA duplex, so less energy is required for separation
84
What does the melting temperature of a nucleic acid show?
The midpoint temperature of transition from double stranded to single stranded forms of nucleic acid (half denaturation point)
85
What is the melting temperature of mammalian genomic DNA?
∼87°C
86
Exactly complementary sequences will hybridise at higher or lower temperatures or salt than sequences which are not exactly complimentary and have base mismatches?
Higher temperatures
| Lower salt
87
What polymerase enzyme is used in PCR?
Taq DNA polymerase
88
What temperatures is PCR performed at?
Denature= 94°C
Anneal=50-60°C
Extend=72°C
89
What length primer should you use for PCR?
About 20 nucleotides for a complex genomic DNA target
90
What base composition should you use in a primer for PCR?
Avoid tandem repeats (can form hair pins)
| %G-C and length should give an equal Tm for each primer (otherwise one may bind without the other)
91
What consideration should you give to the 3' end of a primer for PCR?
Avoid complementarity of the bases at the 3' end. Primer dimers may result causing non-productive amplifications (the primers are then extended instead of the sequence)
Unimportant at the 5' end because polymerisation proceeds 5'-3'
92
What do type II restriction endonucleases do?
Cleave DNA at specific palindromic sequences, usually about 4-8bp long
93
What ends do restriction enzymes produce after cleaving DNA
Blunt of sticky ends
94
Explain the principle of electrophoresis
DNA phosphate backbone has a negative charge, so moves towards the anode (+) when an electrical force is applied.
When forced to move through a porous gel matrix (agarose/polyacrylamide gel) small fragments travel faster than larger ones
After resolution, DNA can be isolated from the gel or transferred to a membrane to form a replica for hybridiation
95
What solid support would target DNA be immobilised on during nucleic acid blotting?
Nylon of nitrocellulose membrane which readily binds single-stranded nucleic acid
96
What would be used to label the probe that will hybridise with the DNA to give exposure on a photographic film during nucleic acid blotting?
Radioactive or fluorescent labelling
97
What is a Southern blot hybridisation?
DNA target and DNA probe
98
What is a Northern blot hybridisation?
RNA target and DNA probe
99
What is a Colony blot hybridisation?
Bacterial DNA target and DNA probe
100
What is a tissue in situ hybridisation?
Chromosome target and DNA probe
101
What is a reverse hybridisation?
Microarrays (Immobilised DNA or oligonucleotide probe, target DNA solution)
102
What is a chromosome in situ hybridisation used for?
Used to locate specific genes on chromosomes using fluorescently labelled DNA probes
