Nucleic Acids and Gene Expression Flashcards
1
Q
What is a nucleic acid?
A
A macromolecule made of a large number of nucleotides.
2
Q
What is the difference between a nucleotide and a nucleoside?
A
Nucleotides are made of a nitrogenous base, a sugar and a phosphate group. Nucleosides only have a nitrogenous base and a sugar.
3
Q
What are DNA and RNA made of?
A
DNA is composed of deoxyribose sugar, a base and a phosphate. RNA has ribose sugar instead. Both are pentose (5 carbon) sugars.
4
Q
What are the 5 nitrogenous bases?
A
Purines: Adenine and Guanine.
Pyrimidines: Cytosine, Thymine and Uracil.
5
Q
What are the nucleosides called?
A
Cytidine, Thymidine, Uridine, Adenosine and Guanosine.
6
Q
What is the structure of DNA?
A
DNA has a right handed double helix structure with two deoxyribose nucleotide strands running antiparallel to each other. The sugar and phosphate form the backbone while the bases face each other in the centre. So there is negative charge on the outside.
7
Q
What is the bonding in the backbone of DNA?
A
The phosphate on the 5’ carbon of deoxyribose is bonded to the -OH on the 3’ carbon of another deoxyribose. This is a phosphodiester bond.
8
Q
What is meant by 5’and 3’?
A
DNA chains are directional. 5’ and 3’ refers to the direction both chains are running. By convention, DNA code is read in the 5’ to 3’ direction.
9
Q
What is the bonding between the chains?
A
The two chains are held together by hydrogen bonds between the bases. Two between A and T (U); three between G and C.
10
Q
What are Watson and Crick base pairs?
A
Each base only hydrogen bond with one other base. Adenine bonds with Thymine (or Uracil in RNA). Guanine bonds with Uracil. This is complementary base pairing.
11
Q
What is the structure of RNA?
A
RNA is a single chain composed of ribose nucleotides. The bonding is the same as DNA.
12
Q
How do you melt DNA?
A
High temperature and/or high salt concentration is used to separate the strands by breaking the hydrogen bonds.
13
Q
What is reannealing of DNA?
A
When the temperature of melted DNA is lowered, the strands will reform into a double helix. This is reannealing.
14
Q
What is the E.coli genome like?
A
E.coli has 4.7 x 10^6 base pairs in a single circular double stranded molecule.
15
Q
What is the human genome like?
A
Humans have 3 x 10^9 base pairs divided into 46 chromosomes that contain linear double helical DNA.
16
Q
How is eukaryotic DNA packaged?
A
DNA is tightly packaged into chromatin which consists of DNA and proteins.
17
Q
What is a nucleosome?
A
Nucleosomes is the lowest level of DNA packaging. It is about 150 base pairs of DNA wrapped around 8 histone proteins.
18
Q
How do nucleosomes form chromosomes?
A
Nucleosomes pack to form fibres which folds into loops which fold further into chromosomes. In the end, the DNA molecule is 10000 times shortened than original extended length.
19
Q
What is the human karyotypes?
A
Humans have 22 pairs of autosomal chromosomes and 1 pair of sex chromosomes.
20
Q
What is meant by semi-conservative replication?
A
Semi-conservative replication means each replicated DNA molecule contains one original strand and one newly synthesised strand.
21
Q
What does DNA polymerase need for DNA replication?
A
A template strand, a primer and free deoxynucleotide triphosphates.
22
Q
What provides the energy for DNA replication?
A
The hydrolysis of deoxynucleotide triphosphates provides the energy for DNA replication.
23
Q
What is the reaction catalysed by DNA polymerase?
A
DNA polymerase catalyses the reaction between the 3’ -OH of the sugar and the phosphate in the next nucleotide. This occurs in the 5’ to 3’ direction.
24
Q
Why can nucleoside analogues be used as drugs?
A
DNA replication needs an exposed 3’ -OH to add further nucleotides. Drugs containing analogue nucleosides are similar to the nucleotides except for the -OH which means replication stops once they are added to the chain. Examples include Acyclovir which is a guanosine nucleoside analogue and can be used as an antiviral drug for herpes.
25
What is the replication fork?
It is a discrete point on the DNA chain where replication begins and proceeds bidirectionally. The hydrogen bonds between the strands are broken by an enzyme called DNA helicase.
26
What is the leading strand?
It is the strand which runs in the 5' to 3' direction. DNA polymerase only works in that direction. So, a primer is added to the beginning at the replication fork and then DNA polymerase completes the replication continuously.
27
What is the lagging strand?
The lagging strand runs in the 3' to 5' direction. Since DNA polymerase can't function in that direction, that strand is replicated in fragments.
28
How is the lagging strand replicated?
When about 200 bp on a lagging strand is exposed, a RNA primer called primase is synthesised by RNA polymerase. DNA polymerase then continues the chain until it meets the next primase. This is repeated throughout the lagging strand to replicate it.
29
What are Okazaki fragments?
The DNA segments in between the RNA primers on the lagging strand is called Okazaki fragments.
30
How are Okazaki fragments joined?
A ribonuclease enzyme (which degrades RNA) removes the primer using 5' to 3' exonuclease activity. DNA polymerase replaces it with new DNA nucleotides. The two fragments are then joined by DNA ligase using ATP.
31
How is accuracy of DNA replication maintained?
DNA polymerase has 3' to 5' exonuclease activity which proofreads the previously added nucleotide before adding a new one. a mistake happens once per 10^9 base pairs. Also, inaccurate RNA primers are replaced by correct DNA.
32
How is E.coli DNA replicated?
E.coli has one circular chromosome. There is only one replication fork and the DNA is replicated bidirectionally until the two forks meet.
33
How is mammalian DNA replicated?
Eukaryotic DNA is very long so there are multiple replication forks along the linear chromosome. Replication ends when all forks meet.
34
What are the stages of the mammalian cell cycle?
Interphase (G1, G0, S and G2); M phase (prophase, metaphase, anaphase and telophase).
35
What occurs during interphase?
During G1 the cell grows and synthesis proteins.
During S phase the DNA is replicated, ready for cell division.
During G2 phase the cell continues growing and synthesises proteins for cell division.
Some cells enter G0 phase where the cell cycle temporarily or permanently stops.
36
What occurs during M phase?
Prophase: DNA condenses into chromosomes and nuclear envelope breaks down.
Metaphase: chromosomes line up along the centre (metaphase plate).
Anaphase: sister chromatids are pulled to opposite poles of the cell.
Telophase: Nuclear envelope reforms around both sets of chromosomes and cell divides.
37
How do chromosomes segregate during metaphase?
Spindle fibres from centrioles attach to the centromere region of the chromosomes. Then, motor proteins pull the them until they all line up in the middle.
38
What is transcription?
This is the process in which the nucleotide information in the DNA is converted to RNA.
39
What is gene expression?
This is when the the information in DNA is used to make RNA which then codes for proteins which are synthesised in the cytoplasm.
40
What are the classes of RNA polymerases and their functions?
RNA Polymerase I: transcribes rRNA genes.
RNA Polymerase II: transcribes genes coding proteins into mRNA.
RNA Polymerase III: transcribes tRNA and 5S RNA genes.
41
What is a gene promoter?
Synthesis of mRNA requires a transcription complex which is assembled during the initiation stage. The DNA sequence this happens at is the gene promoter region.
42
What are transcription factors?
Transcription Factors (TFs) are DNA binding proteins that regulate the amount of transcription from a specific gene.
43
What is a gene promoter composed of?
Gene promoters contain a TF Binding Site to control rate of transcription. They also have a TATA box which is a sequence of Thymines and Adenines that is the initiation point.
44
What is the Basal Transcription complex?
This is the complex of proteins that assemble at the gene promoter to phosphorylate RNA Pol II and begin transcription. Without a TF, it induces a low level of transcription.
45
What is the first step in the assembly of the Basal Transcription Complex?
First TF IID binds to the TATA box. TF IID contains TATA Binding protein (TBP). Once it binds, TF IID, partially unwinds the DNA and widens the minor groove. Since the unwinding is asymmetric, transcription is unidirectional.
46
What are the second and third steps in the assembly of the Basal Transcription Complex?
Secondly, TF IIA and TF IIB bind to TF IID. TF IIB is important because it binds TF IID to RNA Pol II. This is the third stage.
47
What is the final step in the assembly of the Basal Transcription Complex?
The RNA Pol II already has TF IIF bound to it. In the last step, TF IIJ, TF IIE and TF IIH bind to RNA Pol II. TF IIH promotes further unwinding of the DNA helix to allow RNA synthesis.
48
How do transcription factors work?
TFs work by interacting with the DNA and the Basal Transcription Complex to 'bend' DNA to modulate transcription. They can also recruit enzymes to modify histone proteins.
49
How does Aspirin work in relation to TFs?
NFkB is a TF that triggers inflammation by initiating the production of cytokines while IkB is a TF which inhibits NFkB. Aspirin inhibits the breakdown of IkB so NFkB is inhibited, thus preventing inflammation.
50
What is RNA processing?
The RNA made by RNA Pol II is called Pre-mRNA or heterogenous nuclear RNA. It needs to be modified and non-coding parts needs to be removed before it exits the nucleus as mature mRNA. This is RNA processing.
51
What is Pre-mRNA composed of?
The mRNA has exons which has the coding information and it is spread discontinuously in the sequence with introns (non-coding regions) in between.
52
What is a splice donor site?
It is a region near the 5' end of an entron where the splicing of the intron begins.
53
What is a splice acceptor site?
It is a region near the 3' end of the intron where the splicing ends.
54
What sequence do introns begin and end with?
Intron begin with a GU and end with an AG.
55
What is the first stage in RNA processing?
RNA processing uses small Ribonuclear proteins (snRNP's). The first is U1 which binds the splice donor sequence.
56
What is the second stage of RNA processing?
Other snRNP's called U2, U4, U5 and U6 bind to form the spliceosome (the splicing complex). U5 binds to the splice acceptor site. this results in cleavage of the sequence at the splice donor site.
57
What is the branchpoint?
The branchpoint is an A towards the 3' end of the chain which the spliced end from splice donor site binds to through a phosphodiester bond between the 5' phosphate on the G and a 2' OH on the A.
58
What is a lariat structure?
After forming a loop, the phosphodiester bond at the splice acceptor site is cleaved and the exon is removed. It has a lasso shape and is called a lariat structure.
59
What is the sequence at the splice acceptor site?
There are 15 Pyrimidines (U or C) followed by a random base and ending with an AG.
60
What is the RNA cap added?
A methylated guanine called 7-methylguanylate is added to the 5' end of the mRNA to protect it from RNA Endonuclease enzymes and also to enable translation.
61
What is a Poly-A tail?
There is a sequence of AAUAAA in all mRNAs. Polyadenylation happens 11-30 bases downstream from this. A long change of adenines are added to further protect the mRNA.
62
What is Thalassemia?
It is a genetic, haematological disorder in which there is an imbalance in the relative amount of alpha and beta globin chain in haemoglobin.
63
What are the symptoms of Thalassemia?
It causes severe anaemia within the first year of baby's life. It also causes extramedullary hematopoiesis. It leads to hepatic fibrosis and cirrhosis (by the age of 5) and cardiomyopathy.
64
What is the cause of Thalassemia?
There are over a 100 different types of Thalassemia and many of them are due to mutations in the intron sequence of mRNA leading to dysfunctional splicing of the Pre-mRNA.
65
What is Duchenne Muscular Dystrophy (DMD)?
DMD is a severe type of muscular dystrophy which affects 1 in 5000 males (the most common dystrophy). It causes severe muscle weakness from the age of 4 and worsens quickly. Life expectancy is on average 26 years.
66
What is the cause of Duchenne Muscular Dystrophy (DMD)?
The dystrophin gene contains 79 exons. Most DMD mutation cause the deletion of one or more of the exons. This leads to premature abortion of Dystrophin protein synthesis. The protein is important to connect the cytoskeleton of a muscle fibre to the extracellular matrix.
67
What is the C-value paradox?
The complexity of an organism is not always related to its genome size, known as the c-value.
68
What are non-coding RNAs (ncRNA)?
This is any RNA molecule that is not translated into a protein. It includes housekeeping ncRNAs such as rRNA, tRNA and regulatory ncRNAs such as microRNA and siRNA.
69
What is X-inactivation?
This is the process of silencing one of the female X chromosomes so they don't have twice the number of Xs as males. The process is random.
70
How does X-inactivation take place?
X-inactivation is carried out by long ncRNA. It packages one of the X chromosomes in a structure called heterochromatin to make it transcriptionally inactive.
71
Define RNA interference (RNAi).
It is the process by which small RNA molecules inhibit gene expression using microRNAs (miRNA) and small interfering RNAs (siRNA). The process is targeted to direct development and defend against viral nucleotide sequences.
72
What is argonaute?
They are proteins which are the catalytic components of RNA-induced silencing complexes (RISC).
73
What is DICER?
It is an endonuclease that facilitates formation of RISC by cleaving double stranded RNAs (dsRNA) and miRNA into short (20-25 bp) dsRNA fragments with two base overhang at 3' end.
74
What is Drosha?
An enzyme localised in the nucleus which initiates processing of pri-miRNA to form miRNA.
75
What are long ncRNAs?
They are non-protein coding transcripts over 200 nucleotides long and are involved in many regulatory functions.
76
What are miRNAs?
Small (22 nucleotides long) non-coding RNA molecules involved in transcriptional and posttranscriptional gene regulation.
77
What are Piwi-interacting RNAs (piRNA)?
The largest class of small ncRNA molecules (26-31 nucleotides long) and they form RNA-protein complexes by interacting with piwi proteins. They are linked to epigenetic and posttranscriptional silencing in germ line cells.
78
What is pre-miRNA?
They are the substrates for DICER and have a characteristic stem-loop shape of 70 nucleotides.
79
What is pri-miRNA?
A long RNA primary transcript which is cleaved by Drosha to form pre-miRNA.
80
What is RISC?
RISC is a multiprotein complex localised in the cytoplasm which carries out RNAi using miRNA.
81
How does RISC work?
The complex includes a strand of miRNA or siRNA which binds to the complementary mRNA target. This activates RISC RNase leading to cleavage of the mRNA.
82
What are small interfering RNAs (siRNAs)?
A class of dsRNA molecules (20-25 bp long) that are involved in RNAi.
83
What is DNA cloning?
A way to selectively amplify DNA sequences. It can be cell based (in vivo) or cell free (in vitro).
84
What is the first step in cell based DNA cloning?
A target DNA and a replicon (sequence capable of self replication) are cut with specific restriction endonucleases. Then they are mixed with DNA ligase to form recombinant DNA.
85
What is the second step in cell based DNA cloning?
The recombinant DNA is transferred to a host cell (usually bacteria) and it is allowed to grow.
86
What is the final step in cell based DNA cloning?
Once the bacteria multiplies, the plasmid containing recombinant DNA is isolated from the culture to obtain DNA clones.
87
What is selective propagation?
Once the plasmid is transferred to the host, they are grown on an agar plate with antibiotics. Only the bacteria which take up the recombinant DNA have resistance and thus they can be selected for to make the process more efficient.
88
What are Type II restriction endonucleases?
They are enzymes that cleave DNA at specific recognition sequences (usually 4-8 bp). The recognition sites are palindromic sequences. They are part of bacterial immune system.
89
What kind of cleaving do restriction endonuclease enzymes do?
When they cleave DNA, they either produce blunt ends or sticky ends with an overhang to allow binding to other DNA. The length of the cleaved molecules increases as the recognition site length increases.
90
How does DNA electrophoresis work?
DNA molecules are negatively charged so they travel towards the positive charge on a plate. When the molecules travel through the porous gel matrix, the smaller fragments travel faster and thus the molecules separate based on size.
91
What is nucleic acid hybridisation?
A method for detecting specific nucleic acids using a labelled nucleic acid probe.
92
How does nucleic acid hybridisation work?
The target DNA is denatured to form single stranded DNA and then immobilised on a solid support. A complimentary probe, labelled radioactively or fluorescently, is hybridised with it, allowing us to detect the target DNA within the mixture.
93
What is meant by the melting temperature (Tm) of DNA?
It is the midpoint temperature of transition from double stranded to single stranded. It is a measure of nucleic acid stability. Tm for mammalian DNA is 87 degrees celsius. Hybridisation occurs at 25 degrees celsius below Tm.
94
What factors increase DNA melting point?
Strand length - longer strand more hydrogen bonds to break.
Base composition - G-C pairs need more energy than A-T.
Chemical environment - monovalent cations stabilise DNA by neutralising the negative charge.
Denaturants such as urea or formamide destabilise DNA.
95
What is hybridisation stringency?
It is the ability to distinguish between related sequences. High stringency means duplexes only form between strands with perfect complementarity.
96
What factors affect hybridisation stringency?
It increases with an increase in temperature and a decrease in Na+ concentration.
97
What is polymerase chain reaction (PCR)?
PCR is an in vitro process to selectively amplify target DNA within a mixture of DNA sequences.
98
How does PCR work?
The DNA mixture is denatured at 94 degrees celsius and mixed with primers, DNA polymerase and dNTPs. The temperature is lowered to 50-60 to allow annealing between the primer and single stranded DNA. Then temperature is raised to 72 to generate new strands using dNTPs.
99
What is a primer?
A nucleotide sequence (15-25 nucleotides in length. It binds to the DNA strand to form a new complementary strand using dNTPs.
100
Which enzyme is used PCR?
DNA polymerase from Thermophilus aquaticus called Taq polymerase is used because the enzyme is thermostable.
101
What factors are important in primer design for PCR?
Length - about 20 nucleotides to give required specificity to target sequence.
Base composition - avoid tandem repeats to prevent hair pins forming; %GC and length should give same Tm to each primer.
3' end - avoid complementarity of bases at 3' end to prevent primer dimers forming.
102
What is a DNA (oligonucleotide) microarray?
A collection of microscopic DNA spots representing single genes, arrayed on a solid surface.
103
What are microarrays used for?
They are used for mRNA or gene expression profiling. This means they can identify how often a gene is expressed or an mRNA produced. This is useful in comparing normal and cancerous cells for example to detect the difference between them.
104
What is the nature of the genetic code?
The genetic code is read in triplets of bases called codons; it is non-overlapping; the code is degenerate.
105
What are the start and stop codons?
Start codon: AUG (Methionine)
| Stop codons: UAA, UAG and UGA
106
What is the structure of tRNA?
tRNA has a cross shape with three loops. There is an anticodon on one loop and a 3' end with an aminoacyl acceptor. Chain binding is antiparallel like DNA.
107
How does an aminoacyl tRNA form?
First an amino acid is adenylated (AMP added). An aminoacyl tRNA synthetase also bind to it. This ensures the correct amino acid binds to the tRNA, ensuring fidelity of the genetic code.
108
What is the first step in the initiation of translation?
The first step is the assembly of the pre-initiation complex. Met-tRNA binds to eIF2 (eukaryotic initiation factor) with a GTP and to the 40S ribosome unit (only Met-tRNA can do this).
109
What is the second step in the initiation of translation?
eIF4E and eIF4G bind to the 5' cap on the mRNA. The pre-initiation complex recognises this and binds to it.
110
What is the third step in the initiation of translation?
The pre-initiation complex binds to mRNA at the start codon. This sets the frame of translation.
111
What is the final step in the initiation of translation?
The 60S subunit binds to the complex. The GTP is hydrolysed to GDP to ensure correct base pairing. eIF2 and GDP are released. Met-tRNA is bound at the P site on the assembled ribosome with a free adjacent A site.
112
What is the first step in the elongation stage of translation?
Another aminoacyl tRNA binds to the mRNA at the A site. A peptide bond is catalysed between the two adjacent amino acids by peptidyl transferase on the 60S subunit.
113
What is the second step in the elongation stage of translation?
The peptidyl tRNA moves to the P site and the first tRNA disassociates. The two steps of elongation repeats until the stop codon is reached.
114
What is the function of the Elongation factors (EF)?
They are proteins that use hydrolysis of GTP to promote the movement of ribosomes along the mRNA. They increase the accuracy of translation by pausing for hydrolysis to allow incorrect base pairs to dissociate.
115
What happens during termination of translation?
The stop codon is recognised and a release factor (protein) binds to it. Peptidyl transferase hydrolyses the final peptide to release it from the ribosome. Then the release factors and ribosomes dissociate from the mRNA.
116
What are polyribosomes?
Many ribosomes binds to the same mRNA to make several peptide chains.
117
Why do antibiotics inhibit protein synthesis in prokaryotes but not eukaryotes?
Antibiotics are made by bacteria or fungi to give selective advantage over other microbes. Prokaryotes have different translation machinery to eukaryotes such as 70S instead of 80S ribosomes.
118
Where are proteins synthesised?
Proteins that stay in the cell are synthesised in the cytoplasm while extracellular and membrane bound proteins are made in the Rough ER.
119
How are proteins transported to the Rough ER during translation?
The first 20-24 amino acids are a signal sequence (enriched with hydrophobic amino acids) which is recognised by a Signal Recognition Particle (SRP), halting translation. The SRP binds to the SRP receptor on the Rough ER surface and translation resumes. The protein grows into the lumen of the Rough ER.
120
What happens to the secretory protein after translation?
The signal sequence is cleaved by signal peptidase enzymes. Transmembrane proteins have extra hydrophobic sequences that hold them in the membrane.
121
What are the different types of post-translational modification?
Disulphide bond formation; proteolytic cleavage; glycosylation (addition of carbohydrates); phosphorylation (addition of phosphate); prenylation and acylation (addition of phosphate); Hydroxylation.
