DNA and the Flow Of Genetic Information Flashcards
(126 cards)
1
Q
What is amelogenesis imperfecta ?
A
When the enamel is brittle and prone to breakage due to a mutation in a gene
2
Q
Describe the structure of DNA
A
- DNA is a polymer of deoxyribonucleotide units
- the sugar is deoxyribose
- nitrogenous bases include adenine, thymine, cytosine and guanine
3
Q
What is the difference between ribose and deoxyribose ?
A
Ribose has a hydroxyl group on C2 whereas deoxyribose only has a hydrogen atom
- the oxygen in the hydroxyl group of ribose makes it more susceptible to nucleophilic attack so DNA is more stable
4
Q
What are the 2 groups of nitrogenous bases ?
A
- purine - 2 nitrogen containing rings
- pyrimidine - 1 nitrogen containing ring
5
Q
Give some examples of purines
A
- adenine
- guanine
6
Q
Give some examples of pyrimidines
A
- cytosine
- uracil
- thymine
7
Q
What is a nucleoside?
A
A nucleoside contains a purine or pyrimidine bonded to a sugar
- the C1 of the sugar is joined to the N9 of the purine or the N1 of the pyrimidine
- a beta glycosidic linkage is formed
8
Q
Give some examples of nucleosides
A
- deoxyadenosine
- deoxyguanosine
- deoxycytidine
- deoxythymidine
9
Q
What is a nucleotide ?
A
A phosphate ester of a nucleoside
- nucleoside with a phosphate group attached by a phosphate ester bond
10
Q
Give some examples of nucleotides
A
- deoxyadenosine 5’ - triphosphate (ATP)
- deoxyguonosine 5’ - triphosphate
11
Q
Describe the structure of the DNA polymer
A
- the DNA backbone contains deoxyribose linked by phosphate groups (this section is invariant)
- the variable part is the sequence of the 4 bases
- the DNA chain has polarity
12
Q
Why does the DNA chain have polarity
A
- the deoxyribose sugar is linked to the phosphate group by 3’ 5’ phosphodiester bonds
- there is a 5’ end on the chain where there is a free phosphate group
- there is a 3’ end where there is a free hydroxyl group
13
Q
Describe the structure of RNA
A
- unbranched polymer of nucleotides
- the sugar is ribose
- there is no thymine base but rather uracil
- has polarity
- usually single stranded but may contain secondary structures
14
Q
What is the most common RNA secondary structure ?
A
The hairpin loop
15
Q
Who discovered the double helical structure of DNA ?
A
James Watson and Francis Crick
16
Q
What are the key features of the double helix of DNA ?
A
- there are 2 helical polynucleotide chains coiled around a common axis
- the 2 chains are anti parallel (run in opposite directions)
- the bases are found on the inside of the helix
- the phosphate and deoxyribose units are found on the outside
- the planes of the bases are perpendicular to the helix axis
- the 2 chains are held together by hydrogen bonds
17
Q
Describe base pairing in DNA
A
- a purine base pairs with a pyrimidine
- adenine base pairs with thymine using 2 hydrogen bonds
- cytosine base pairs with guanine using 3 hydrogen bonds
- the sequence of bases varies and carries the genetic information
18
Q
What is meant by melting temperature ?
A
The temperature at which DNA goes from a double strand to a single strand
19
Q
Why does the melting temperature of DNA vary ?
A
DNA with an abundance of cytosine and gaunine needs a higher temperature to break the hydrogen bonds because there are 3 bonds
DNA with an abundance of adenine and thymine needs a lower temperature to break the hydrogen bonds because there are 2 bonds
20
Q
Describe the structure and packaging of DNA in prokaryotes
A
- circular double stranded molecule of DNA
- it is twisted upon itself to form supercoiled DNA
- supercoiled DNA is located in the region of the cell called the nucleoid
21
Q
Describe the structure and packaging of DNA in E.Coli
A
- it is a circular double stranded DNA molecule
- it is twisted upon itself to form supercoiled DNA
- it is located in the region of the cell known as a nucleoid
- the supercoiled DNA is organised into 50 or so loops that is bound to a central protein core which is attached to the cell membrane
22
Q
Which nucleic acids can be present in viruses and how are they organised ?
A
- RNA
- DNA - can be linear, circular or both
23
Q
Give an example of a virus that has linear DNA
A
T7 bacteriophage
24
Q
How is eukaryotic DNA packaged ?
A
- eukaryotic DNA is linear and the large size requires packaging
1) DNA is tightly bound to positively charged histone proteins to form nucleosomes
2) the nucleosome core contains 140 base pairs of DNA wound around a histone octane (2x H2A, H2B, H3 and H4) and linker DNA contains 60 base pairs of DNA bound to histone 1 to give a packing ratio of 7 (this is known as the beads in string model)
3) nucleosomes are coiled into a helical array to form 30 nm chromatin fibres to give a packing ratio of 40 - there are 6 nucleosomes per turn of helix
4) fibres are folded into loops and stabilised by non histone proteins to give a packing ratio of 100 to 10000
25
Which histones are involved in the packaging of DNA ?
- H1
- H2A
- H2B
- H3
- H4
26
What is a nucleosome ?
DNA tightly bound to a histone
27
What is the semiconservative model of DNA replication ?
Each strand acts as a template for the synthesis of a new strand
28
What are the 3 models of DNA replication ?
- semiconservative
- conservative
- dispersive
29
Which enzyme catalyses the process of semi-conservative replication ?
DNA polymerase
30
What does DNA polymerase require in order to function ?
- DNA template containing single stranded regions (substrate)
- all four deoxynucleotidetriphosphates : dATP, dGTP, dTTP, dCTP
- Mg2+ which is a cofactor
- a short piece of RNA primer with a 3’ - OH group
31
How does DNA polymerase work ?
Synthesises in the 5' to 3’ direction
32
How many DNA polymerases are found in E.coli and what are they ?
There are 3 -
- DNA polymerase I
- DNA polymerase II
- DNA polymerase III
33
What is 5' to 3' synthetic activity and which of the DNA polymerases found in E.coli can do this ?
- when the RNA primer with the 3’ - OH group is used to add dNTPs
- all 3 DNA polymerase scan do this
34
What is 3' to 5' exonuclease activity and which of the DNA polymerases found in E.coli can do this ?
- it is the process of proof reading by the erasing of bases
- all 3 DNA polymerase scan do this
35
Which of the DNA polymerases found in E.coli can do 5’ to 3’ exonuclease activity ?
Only DNA polymerase I
36
What are DNA polymerase I and III involved in ?
DNA replication
37
What is DNA polymerase II involved in ?
DNA repair
38
Describe the process of DNA replication in eukaryotes
- an RNA primer and DNA polymerase III enzyme synthesise a continuous strand of DNA known as the leading strand in the 5’ to 3’ direction (one primer is needed)
- this cannot happen in the 3’ to 5’ direction and so a lagging strand of Okazaki fragments form (more than one primer is needed)
- DNA polymerase I removes sections of the RNA primer and replaces them with DNA nucleotides
- the strands are joined together by DNA ligase
39
What are Okazaki fragments ?
Short sequences of DNA nucleotides which are synthesised discontinuously and linked by DNA ligase
40
Which DNA polymerase enzyme is used to remove the RNA primers ?
DNA polymerase I because 5’ to 3’ exonuclease activity is needed
41
What is the role of DNA ligase ?
Joins the fragments of newly synthesised DNA to form a seamless strand
42
Describe the process of DNA replication in prokaryotes
1) there is a single origin of replication known as an OriC locus which binds DnaA protein (origin recognition protein)
2) several proteins join in including DNA B which is a helicase
This causes the double helix to unwind using ATP as an energy source
3) the protein DNA topoisomerase I allows the helix to unwind without causing extensive rotation of the chromosome
4) the unwound portion of DNA is stabilised by single strand binding proteins (SSB)
5) DNA synthesis is primed by an RNA primer
The primer is ~ 5 nucleotides and formed by the primase enzyme
6) both the leading strand and the Okazaki fragments in the lagging strand are then synthesised by DNA polymerase III
7) the RNA primer is then hydrolysed by the 5’ to 3’ exonuclease activity of DNA polymerase I
8) the Okazaki DNA fragments are joined by DNA ligase
9) topoisomerase II enzymes separate the 2 daughter DNA circles
43
Summarise the different proteins involved in DNA replication
• helicase (dnaB) - unwinds the double helix
• primase - synthesises RNA primers
• SSB - stabilises single stranded regions
• DNA topoisomerase I - allows helix unwinding without extensive rotation of the chromosome
• DNA polymerase III - synthesises DNA
• DNA polymerase I - erases primer and fills in gaps
• DNA ligase - joins the ends of DNA
• DNA topoisomerase II - separates the 2 DNA circles
44
Describe the differences between eukaryotic and prokaryotic DNA replication
- in eukaryotes DNA replication occurs semiconservatively and bidirectionally from several thousand origins in the synthesis phase
- eukaryotic cells contain several types of DNA polymerases
- in eukaryotic cells, old histones stay with the leading strand and new histones assemble on the lagging strand
45
Describe the replication of an RNA virus
- reverse transcriptase uses viral RNA to make a DNA-RNA hybrid
- the reverse transcriptase then erases the RNA strand
- the reverse transcriptase enzyme then uses the DNA strand as a template to form double helical viral DNA
46
What is the key enzyme involved in the replication of an RNA virus ?
Reverse transcriptase
47
What is transcription ?
When a DNA molecule is transcribed into a RNA molecule
48
Summarise the different types of RNA found in a cell
- messenger RNA (mRNA)
- ribosomal RNA (rRNA) : part of ribosomes
- transfer RNA (tRNA)
- small nuclear RNA (snRNA) : involved in the processing of pre-mRNA
- microRNA (miRNA)
- small interfering RNA (siRNA)
- small nucleolar RNA (snoRNA)
- long non coding RNA (lncRNA) : >200 nucleotides
- catalytic RNA (ribozymes)
49
Briefly summarise mRNA
- it is the RNA that carries information from DNA to the ribosomes in the cell
- the mRNA code sequences determine the amino acid sequence in the protein that is produced
50
Briefly summarise tRNA
used to transfer specific amino acids to growing polypeptide chains at the ribosomal site of protein synthesis during translation
51
Briefly summarise miRNA
- used to regulate gene activity
- they are small (~22 nucleotides) RNA molecules that regulate the expression of mRNA molecules in particular pathways
52
Briefly summarise siRNA
- similar to miRNA but generally have more specific binding targets
53
Briefly summarise snoRNA
Guides chemical modifications of other RNAs
54
Briefly summarise catalytic RNA
Functions as an enzymatically active RNA molecule
55
What is a sense strand ?
Strand of DNA which is identical to mRNA but in mRNA there are uracil bases instead of thymine bases
56
What is an anti sense strand ?
Strand of DNA which is complementary to mRNA and is used as a template
57
How many types of RNA polymerase are there in eukaryotes ?
There are 3 types
58
How many types of RNA polymerase are there in prokaryotes ?
1 type
59
Briefly describe the function of RNA polymerase
- catalyses transcription
- no proofreading activity
- no primer required
60
Describe how RNA polymerase works
- synthesises RNA according to instructions from the DNA template
- the activated monomer substrates are ribonucleoside triphosphates (ATP, GTP, UTP and CTP)
- synthesis of RNA occurs in the 5’ to 3’ direction
61
Describe the RNA polymerase structure in prokaryotes
A 5 polypeptide chain containing 2 alpha units, 1 beta unit, 1 beta prime unit and 1 omega unit known as a core enzyme binds with a sigma subunit and this forms the RNA polymerase holoenzyme
62
Where does RNA polymerase bind ?
the sigma subunit within RNA polymerase recognises the promoter sequence on the DNA molecule and takes the RNA polymerase to this region where it can bind
63
What are the 3 stages of transcription ?
- initiation
- elongation
- termination
64
Describe the initiation stage of transcription
The sigma subunit within the RNA polymerase holoenzyme recognises the promoter sequence of DNA and fixes the RNA polymerase enzyme at this site
65
Describe the elongation stage of transcription
1) local unwinding of the DNA double helix
2) the sigma unit dissociates from the RNA polymerase enzyme
3) RNA is synthesised by RNA polymerase in the 5’ to 3’ direction
66
Describe the termination stage of transcription
- occurs when a stop signal indicates that the end of a gene is encountered
- stop signals include :
• formation of an RNA secondary structure
• addition of Rho factor onto RNA polymerase and energy from ATP
67
What are the post transcriptional modifications ?
- splicing of introns (specific to eukaryotes)
- addition of a 5’ CAP of 7-methylguanylate and a 3’ poly(A) tail (specific to eukaryotes)
- cleavage of primary transcripts to generate several RNA species e.g. tRNA and rRNA
- addition of nucleotides to the ends of some RNA chains
- RNA editing
- modifications of bases and ribose units
68
Describe the splicing of introns
- occurs in the nucleus
- introns in pre-mRNA are cut out and the exons join together
- occurs at splicing sites where there are conserved sequences which are recognised by splicing machinery
- alternative splicing increases the versatility of eukaryotic genes and generates different proteins from a single gene
69
Describe process and purpose of the addition of a 5’ CAP of 7-methyguanylate and a 3’ poly(A) tail
- the 5’ end of mRNA is linked to a modified G base by phosphate groups
- the 3’ end contains ~ 200 A bases known as a poly(A) tail which adds stability to the mRNA molecule
70
Where does translation in eukaryotes take place ?
In the cytoplasm
71
What is the relationship between the synthesis of polypeptide chains and the translation of mRNA ?
- polypeptide chains are synthesised from the N to the C terminus
- mRNA is translated from the 5’ to 3’ direction
72
What is the genetic code ?
- amino acids are coded by a group of 3 bases known an a codon
- the genetic code is non overlapping
- the genetic code is degenerate which means some amino acids have more than one code
- it is the same in most organisms
73
Describe what is meant by the genetic code being degenerate
- there are 20 amino acids so 64 possible combinations
- 61 specify for amino acids
- 3 stop codons (UAA, UGA and UAG)
74
What is the only code for methionine ?
AUG
75
What is an anticodon ?
A set of 3 bases on a tRNA molecule which is complementary to a codon
76
What is the role of tRNA ?
- carries amino acids in an activated form
- there is a specific tRNA molecule for each amino acid
77
Is the entire mRNA molecule translated ?
- not all of the sequence is translated
- the open reading frame (ORF) of the mRNA molecule is the only section that is translated
- the untranslated regions (UTR) are not translated but they may contain sequences which are used for other things e.g. the 5’ end UTR contains sequences which regulate translation
78
Describe the structure and function of tRNA molecules
- carry activated forms of amino acids in translation
- activated amino acid precursors are aminoacyl-tRNAs
- the addition of amino acids to the tRNA molecule is catalysed by the enzyme aminoacyl-tRNA synthetase or activating enzyme
- for each amino acid there is at least one tRNA and activating enzyme which gives specificity in the process of translation
79
Where does protein synthesis occur ?
On ribosomes in the cytoplasm
80
Describe the structure of 70s ribosomes
- 70s ribosomes can be split into a 50s subunit (large) and 30s subunit (small)
- 50s subunit contains 32 proteins which contains 23s rRNA and 5s rRNA
- 30s subunit contains 21 proteins which contains 16s rRNA
81
What are the 3 sites on a ribosome ?
- E site (empty) which binds tRNA prior to release from ribosomes
- P site (peptidyl) which is where tRNA linked to a growing polypeptide chain is bound
- A site (Aminoacyl) which is where incoming aminoacyl-tRNA binds
82
What are the 3 stages of translation ?
- initiation
- elongation
- termination
83
Describe the process of initiation in translation
- the Shine-Dalgarno sequence recognises 16s rRNA and so the mRNA binds to the small subunit
- the complementary anticodon of initiator tRNA binds to the AUG start codon
- the large subunit comes in using energy from GTP
- this forms a translation initiation complex
- tRNA carrying a N-formylmethionine amino acid aligns at the P site of the ribosome
84
Describe the elongation phase of translation
- energy from GTP is used to bind another tRNA molecule carrying a second amino acid to the next codon on the A site of the ribosome
- enzyme peptidyl-transferase forms a peptide bond between the 2 amino acids
- translocation occurs and the ribosome moves to the next codon
- the first tRNA molecule is released and this binds to the E site
- a 3rd tRNA molecule binds to the A site and the whole process continues until a stop codon is reached
85
Describe the termination phase of translation
- when a stop codon is reached the peptidyl-transferase enzyme is altered
- the polypeptide chain is hydrolysed and released when the protein is finished in translation
86
What are the differences between translation in prokaryotes and eukaryotes ?
- in eukaryotes there are 80s ribosomes
- in eukaryotes the initiating amino acid is methionine and not N-formylmethionine
- in eukaryotes there is no Shine-Dalgarno sequence
- there are differences in initiation, elongation and termination factors
87
How is the first AUG sequence identified in eukaryotes ?
There is scanning machinery which binds to the 5’ CAP and starts scanning the mRNA molecule to find the first AUG codon (requires energy from ATP)
88
Why do antibiotics work ?
Antibiotics are effective in killing bacteria because of differences in translation between prokaryotes and eukaryotes
89
How do proteins end up in different parts of the cell if protein synthesis takes place in the cytoplasm ?
Proteins contain signals which are recognised by machinery which takes them where they are supposed to be
90
List some post translational modifications
- disulphide bond formation between cysteines
- addition of a prosthetic group
- glycosilation of proteins
- acetylation of the N terminus
- hydroxylation of proline
- carboxylation of glutamic acid
- addition of a fatty acid chain
- phosphorylation or dephosphorylation
- cleavage or trimming of proteins after synthesis
91
Give an example of the addition of a prosthetic group to a protein
The addition of a haem group to globin in haemoglobin
92
What does glycosilation do to proteins ?
The addition of sugars to proteins makes them hydrophilic
93
What does the addition of a fatty acid chain do to proteins ?
It makes proteins hydrophobic
94
What is a gene ?
A gene is a DNA region that may be transcribed into mRNA
95
What is an operon ?
Several related structural genes with a single promoter region and a shared operator region
96
How are operon genes transcribed ?
They are transcribed together into polycistronic mRNA
97
How is gene expression regulated in bacteria ?
- positive control mechanisms stimulate gene expression
- negative control mechanism inhibit gene expression
98
How do positive control mechanisms work ?
If an activator binds to the activator binding site then RNA polymerase binds to the promoter region and transcription takes place
99
How do negative control mechanisms work ?
When a repressor protein binds to the operator region then no transcription takes place
100
What are activators and repressors ?
They are proteins which are coded for by constitutive regulatory genes elsewhere in the genome
101
What are the 2 types of operons ?
Operons are either repressible or inducible
102
What are repressible operons ?
- involve the binding of a co-repressor
- this turns off operon transcription
103
What is an inducible operon ?
- involves the binding of an inducer
- this turns on operon transcription
104
What is the Lac Operon ?
A group of genes that encode proteins that allow bacteria to use lactose as an energy source
- the Lac operon is involved in the digestion of lactose
105
What is the function of the Lac operon of E. Coli ?
It makes the 3 enzymes required for the breakdown of lactose -
- the LacZ gene codes for the enzyme beta-galactosidase
- the LacY gene codes for the enzyme permease
- the LacA gene codes for the enzyme transacetylase
106
What is the function of the enzyme beta-galactosidase in the breakdown of lactose ?
- breaks down lactose into glucose and galactose
- isomerises lactose into allolactose inducer
(if lactose is present then allolactose is also present)
107
What is this function of the enzyme permease in the breakdown of lactose ?
Important for lactose transport across the cell membrane
108
What are the 2 control circuits of the Lac operon ?
- negative inducible
- positive inducible
109
Describe the negative inducible control mechanism of the Lac operon when lactose is not present
1) the LacI gene produces mRNA
- this mRNA produces the Lac repressor protein
2) the Lac repressor has a higher affinity for the operator region compared to the affinity of RNA polymerase for the promoter region
3) so the Lac repressor protein binds to the operator region
4) RNA polymerase cannot bind to the promoter region and so no RNA is made for the genes of the enzymes which breakdown lactose
110
Describe the negative inducible control mechanism of the Lac operon when lactose is present
1) the LacI gene produces mRNA
- this mRNA produces the Lac repressor protein
2) Allolactose (isomer of lactose) binds to the Lac repressor and so it cannot bind to the operator region
3) RNA polymerase binds to the promoter region and so the genes for the enzymes are transcribed
111
Describe the positive inducible control mechanism of the Lac repressor
- when glucose levels are low cyclic AMP (cAMP) is produced from ATP
1) cAMP binds to a CAP protein which binds to the Lac operon and increases the affinity of RNA polymerase to the promoter region
2) this increases the transcription of the genes
112
In what circumstance will there be the highest transcription of Lac operon mRNA ?
- no glucose present so high levels of cAMP
- lactose is present
113
What is genomics ?
The study of an organisms genome
114
What is transcriptomics ?
The study of the transcriptome (the expressed RNA)
115
What is proteomics ?
The study of the proteome (proteins that are synthesised)
116
What is metabolomics ?
The study of the small molecule components of a cell
117
What are the components required for the production of recombinant DNA molecules ?
- restriction enzymes
- reverse transcriptase
- vector
- DNA ligase
- a host for producing large amounts of recombinant DNA molecules
118
Describe the role of restriction enzymes
They cut at a specific sequence to produce sticky ends which are complementary
- the same restriction enzyme can be used to cut bacteria and human DNA
119
How are recombinant DNA molecules generated ?
1) restriction enzyme cuts the DNA
2) the vector and DNA have complementary sticky ends
3) DNA ligase joins the complementary bases
120
Give an example of a naturally occurring vector
Plasmid
121
What is agarose gel electrophoresis ?
A technique used to separate DNA according to its size from the negative end to the positive end
122
What is polymerase chain reaction used for ?
It is used to make large amounts of DNA
- growth is exponential
123
Describe the process of PCR
1) the template DNA is heated to 95°C causing the 2 strands to separate
2) the temperature is lowered and the DNA primers anneal to the target sequence
3) Taq polymerase allows new DNA to be synthesised
124
What are the applications of PCR ?
- quick isolation of some genes without the need to screen libraries
- detection of bacterial and viral infections
- identifying genetic disorders
- early detection of certain cancers that are due to mutation of specific genes
- monitoring therapy
- sex determination of prenatal cells
- forensics and legal medicine
- genetically modified food detection
- wildlife conservation
125
What are some other molecular biology techniques ?
- DNA sequencing
- in situ hybridisation
- inhibition of expression of specific genes by use of anti-sense RNA / synthetic DNA and siRNA
- generation of knock-out mice
- cloning of animals
- gene expression profiling
- RNA sequencing
- gene editing
126
What is the application of molecular biology techniques ?
- basic research
- large scale production of recombinant proteins for medical use e.g. insulin
- over expression of specific genes in transgenic animals or plants
- mapping and cloning human disease genes
- DNA based diagnosis of genetic diseases
- gene therapy
