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3: Genes Flashcards

1
Q

What are the primary properties of genetic material?

A

Must have information content
Must be very stable but also flexible
Must be subject to mutation (for evolution)
Must be able to make copies in an error-free way
Must be able to retrieve the information content

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2
Q

What are DNA nucleotides made up of?

A

Phosphate group
Pentose sugar (deoxyribose joined to phosphate by the 5’ carbon)
Nitrogenous base (joined to the 1’ carbon of the deoxyribose sugar)

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3
Q

What are the two types of nitrogenous bases?

A

Purines: adenine and guanine
Pyrimidines: cytosine and thymine

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4
Q

Describe the properties of DNA.

A

A polynucleotide chain
Sugars joined by a 5’ to 3’ phosphodiester linkage
DNA has a hydrophilic pentose-phosphate ‘backbone’
The free 5’ OH is the 5’ end of the chain (the start)
The free 3’ OH is at the 3’ end of the chain (the end)

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5
Q

What is Chargaff’s Rule?

A

[A] = [T]
[C] = [G]

E.g. in human DNA,
30%:30% adenine and thymine
20%:20% cytosine and guanine

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6
Q

Describe the 3D structure of DNA.

A

Antiparallel, right-handed double helix
Bases stacked in pairs on top of each other

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7
Q

How do bases on opposite DNA strands join?

A

Via hydrogen bonding
Complementary base pairs of A-T and C-G

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8
Q

What makes the DNA double helix so stable?

A

Hydrogen bonds between base pairs
Hydrophilic effect of bases expelling water from the centre
Van der Waals forces between adjacent (stacked) bases
Hydrophilic interactions of polar phosphate and water on the outside

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9
Q

How are DNA sequences written?

A

As the two strands
The top strand is always 5’-3’
Usually abbreviated to the first strands sequence, 5’-3’ is usually assumed

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10
Q

What is B-Form DNA?

A

Most common form of DNA
Backbone on the outside
Bases inside
Bases nearly perpendicular to the helical axis
Adjacent bases separated by 0.34nm
Repeated unit every ~10 bases
Major and minor grooves, important for protein binding

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11
Q

What are the differences between RNA and DNA?

A

RNA is less stable than DNA
Major difference is that RNA is made of one polynucleotide chain rather than two like in DNA
Also the pentose sugar is ribose rather than deoxyribose

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12
Q

Describe how DNA strands can serve as a template for DNA replication.

A

DNA synthesis is progressive and bidirectional
Spreads away from the origin point in both directions

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13
Q

How many hydrogen bonds are there between A-T and C-G?

A

A-T: 2
C-G: 3

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14
Q

What are NTPs?

A

Nucleoside triphosphates
Building blocks of nucleic acids like DNA and RNA and energy sources like ATP and GTP

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15
Q

What is the difference between an NTP and a nucleotide?

A

Nucleotide: base + sugar + at least one base
Nucleoside triphosphate: base + sugar + three phosphates

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16
Q

What is dNTP?

A

Deoxyribonucleoside triphosphate, and it is the building block used for DNA synthesis. Examples include dATP, dGTP, etc. It is used by DNA polymerases to build new DNA strands during replication and repair. Energy from breaking the phosphate bonds helps to drive the reaction.

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17
Q

What is the major replication enzyme for DNA?

A

DNA polymerase III

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18
Q

Describe the function of DNA polymerase III.

A

Major replication enzyme
Requires a template strand to guide the new strand assembly by complementary base pairing
Requires dNTPs
Attaches nucleotides to the 3’ end of the new strand so synthesises in the 5’-3’ direction
Releases two phosphates (pyrophosphates)

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19
Q

What is semi-discontinuous replication?

A

The process of DNA synthesis on the two strands
DNA synthesis is continous on the leading strand (5’-3’) but lags on the other strand
The lagging strand is synthesised in shorter fragments “Okazaki fragments”
The gaps in the fragments are sealed via DNA ligase
This overcomes the issue of DNA polymerase III only working in a 5’-3’ direction

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20
Q

What evidence is there for semi-discontinuous replication?

A

The presence of short DNA fragments in DNA extracted from Okazaki fragments
These short fragments are not found if DNA synthesis is stopped by inhibitory drugsc

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21
Q

What is transcription?

A

The process by which a cell makes an RNA copy of a segment of DNA

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22
Q

Describe the process of transcription.

A

1) DNA is ‘unzipped’ via action of RNA polymerase II which binds to a specific promoter region of DNA and separates the strands
2) RNA poylmerase II moves along the DNA template strand and builds a complementary mRNA strand using the DNA as a template
3) The mRNA strand is then released when the end of the gene is reached by RNA polymerase II

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23
Q

What are the three main classes of RNA?

A

mRNA (messenger RNA)
tRNA (transfer RNA - involved in protein synthesis, or translation)
rRNA (ribosomal rNA, major component of ribosomes)

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24
Q

Describe how the mRNA strand produced relates to the coding strand.

A

The mRNA strand produced during transcription will be complementary to the non-coding strand
The non-coding strand will have a complementary sequence to the coding strand
Therefore, if the non-coding strand is used as a template, the synthesised mRNA will have an identical sequence to the coding strand
The only difference will be that thymine is replaced by uracil

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25
Why is transcription necessary as an intermediate step of protein synthesis?
1) Amplification of genetic information by making lots of copies of mRNA 2) Compartmentation: allows DNA to be copied into mRNA which can then be exported into the cytoplasm to make proteins - DNA can’t leave the nucleus and therefore remains protected inside 3) Easier to use and control the 'working copy' of RNA without compromising the 'master copy' of DNA
26
Describe RNA Polymerase II.
One of three types of RNA polymerase Requires a DNA template Requires activated precursors, ATP, GTP, CTP and UTP Synthesises in the 5' to 3' direction Does not require a primer High error rate due to no proof reading activity
27
What is the role of NTPs in transcription?
NTPs are the raw materials used by RNA polymerase II to build the RNA strand ATP, CTP, GTP, and UTP each correspond to a base Two of the phosphates on the NTP are cleaved off (the beta and gamma phosphates) and this hydrolysis releases the energy for a phosphodiester bond to form between the 3' hydroxyl of the previous nucleotide and the 5' phosphate of the incoming one (the alpha phosphate of the NTP) The beta and gamma phosphates released are pyrophosphate (PPi)
28
What are the coding and non-coding strands in transcription?
Coding strand = sense strand Non-coding strand = nonsense strand, ot antisense strand
29
How does the speed of transcription compare to the speed of DNA replication?
Transcription is much slower RNA polymerase needs transcription factors to help bind RNA polymerase, where DNA polymerase does not Splicing has to occur during transcription, as well as capping and polyadenylation, but DNA replication doesn’t require any modification processes DNA polymerase is a faster enzyme DNA polymerase has more robust error-checking capabilities ensuring replication fidelity at high speeds
30
What is the sigma factor?
A subunit of bacterial RNA polymerase that helps the enzyme to recognise and bind to the promoter sequences at the start of transcription It dissociates after transcription begins, allowing RNA polymerase to continue elongating RNA
31
What is the promoter region?
The DNA sequence where transcription begins and RNA polymerase II binds Bacteria and eukaryotes have different promoters
32
What is the +1 nucleotide?
The first nucleotide to be transcribed Any nucleotide upstream (before this) is assigned a negative number
33
What is the TATA box?
A sequence at the -10 position that is a critical part of the promoter region, and helps RNA polymerase know where to bind and start transcription Humans also have a TATA box but not in the same position, rather it is around 25-30 nucleotides upstream of +1
34
What is the -35 element?
A sequence recognised by the sigma factor helping RNA polymerase identify where to begin transcription (unique to bacteria)
35
Why are genes transcribed individually?
Each gene has its own promoter and regulatory elements that control when and how much of the gene is transcribed, allowing for precise control of gene expression in different conditions or cells types
36
How does quantity of transcription vary for different genes?
Varying cell types will have different protein requirements, e.g. might need more structural proteins, etc. They will vary the amount of transcription they undergo for each gene depending on how much of each protein they need
37
At what quantity are the genes for enzymes transcribed in most cells?
Usually in quite low quantities Because enzymes can be reused over and over again Therefore they don't need to be rapidly resynthesised in high quantities
38
What happens to pre-mRNA after transcription?
1) Processing 2) Nuclear export (via nuclear pores) 3) Protein synthesis 4) mRNA degradation
39
What RNA processing do eukaryotes undergo after pre-mRNA is synthesised?
Capping Polyadenylation Splicing
40
What is capping?
Where a nucleotide is attached to the 5' end of the strand Known as the '5' cap' This protects the 5' end from nucleases, makes it stable, and acts as a label to signal translation to begin
41
What is polyadenylation?
Where a long stretch of adenine is added to the 3' end of the newly formed mRNA by poly(A) polymerase This creates a poly-A tail, which adds stability to that end of the mRNA
41
What is splicing?
The process by which introns are cut out of pre-mRNA and exons are spliced together This is done by spliceosomes
41
What are spliceosomes?
Large complexes made up of smaller small nucleoproteins (snRNPs) and other proteins It recognises the splice sites at the junction of introns and exons, removing introns and joining exons together
42
What is pre-mRNA?
The form of mRNA synthesised from transcription before any modifications have occurred
43
Describe how mRNA is degraded.
mRNA will be digested by ribonuclease enzymes These break the phosphodiester bonds between individual ribonucleotides This is a good way to control and regulate protein synthesis
44
What is translation?
The process of making DNA from mRNA mRNA
45
What is a codon?
A group of three bases Forms the triplet code
46
How is the genetic code degenerate?
Multiple codons code for the same amino acid
47
Which codons are the stop codons?
UAA UAG UGA These do not code for an amino acid, they tell the system where the polypeptide chain should end
48
Which codon is the start codon?
AUG Codes for methionine in eukaryotes
49
How is the genetic code universal?
It applies to all organisms
50
Describe how the reading frame works in translation.
Codons are read sequentially from the 5’ to 3’ direction AUG marks the start of the open reading frame (ORF) and codes for methionine, so all proteins begin with methionine In eukaryotes, the start codon is usually the first AUG downstream of the mRNA cap In prokaryotes, the start codon is usually the first AUG downstream of the Shine-Dalgarno sequence (AGGAGG) Stop codons UAA, UGA, and UAG signal the end of translation when they appear in the correct reading frame
51
What is the Shine-Dalgarno sequence?
AGGAGG In prokaryotes, the start codon for translation is usually the first AUG downstream of this sequence
52
Describe the anatomy of mRNA.
Has a 5' cap, then the 5' untranslated region Then the ORF (open reading frame) Then the 3' untranslated region, capped with a poly A tail
53
What are indels?
Insertions and deletions of one or more nucleotides in the gene
54
What are point mutations, and give three examples.
Substitutions Missense: codon changes and leads to the corresponding amino acid changing Silent: codon changes but makes the same amino acid due to degeneracy Nonsense: codon changes to a stop codon (e.g. UAA)
55
What is a frameshift mutation?
Causes the sequence to shift either to the left or the right, therefore affecting the entire open reading frame rather than just one codon
56
What are ribosomes?
Complex structure made up of a large and small subunit Site of translation
57
What is tRNA?
An adaptor molecule that is used to 'read' the transcript Clover leaf structure due to hydrogen bonding folding in on itself 3' end is attached to the amino acid Anticodon is complementary to the mRNA codon Each amino acid/tRNA combination has a specialised enzyme to attach to the amino acid (aminoacyl tRNA synthetases)
58
What is the name of the enzyme that attaches tRNA to its amino acid?
Aminoacyl tRNA synthetases
59
Describe the elongation cycle.
Ribosomes have three tRNA docking sites (E, P, and A) and an mRNA binding site 1) aminoacyl-tRNA enters the A site, guided by its complementary anticodon 2) The amino acid in the P site is transferred and bonded to the one in the A site, lengthening the polypeptide (catalysed via peptidyl transferase) 3) The ribosome shifts along the mRNA, so the tRNA in the P site moves to the E site and exits 4) The tRNA in the A site now moves into the empty P site 5) The A site is then free for the next aminoacyl-tRNA to join
60
What is termination?
When translation ends because of stop codons There is no tRNA that can bind to a stop codon because it does not code for an amino acid Therefore release factors bind to stop codons and cause termination A molecule of water is added to the polypeptide which is then released to the mRNA strand
61
What are each of the ribosome docking sites for?
A: arrival of aminoacyl-tRNA P: peptide bond formation E: exit of the now-empty tRNA
62
Why is the double helix of DNA so stable?
Stabilised by hydrogen bonds Which can also be broken in correct conditions, enabling DNA replication to occur
63
What detergent can you use to break down cell membranes in preparation of precipitation of nucleic acids?
Sodium dodecyl sulphate (SDS)
64
Why is DNA negatively charged?
Because of the presence of phosphate groups within the sugar phosphate backbone
65
What is the hyperchromic effect?
An increase in UV absorbance at 260nm when DNA denatures and separates into two strands This is because double stranded DNA absorbs less UV light due to base stacking and hydrogen bonds Base stacking constricts the ability of electrons to absorb photons
66
What is Tm in DNA melting curve?
The temperature at which 50% of the DNA is denatured This depends largely on the base proportions of DNA If rich in GCs, the melting temperature will be much higher because there are 3 hydrogen bonds between G and C, but only 2 for A and T
67
What is gel electrophoresis?
Separates DNA fragments by size, smaller ones move faster through (in a sieving effect) agarose/polyacrylamide gel towards the positive electrode Ethidium bromide dye intercalates into DNA and fluoresces under UV light, so creates tracks Alternatively, can use radioactive labelling with P32 during in vitro replication Produces an autoradiograph which is exposed to photographic film to make tracks This can be used in forensics
68
What are restriction endonucleases?
Enzymes from bacteria that cut DNA at very specific recognition sites These sites are usually a 6 base pair sequence
69
What are recognition sequences?
The sites at which restriction endonuclease enzymes bind to cut Most recognition sequences are palindromic, because this allows the enzyme to bind symmetrically and cut both strands at the same position
70
How can you detect specific fragments after gel electrophoresis?
Southern blotting Or PCR
71
What is Southern blotting?
DNA digested with restriction endonucleases and run on a gel to produce a smear of fragments DNA in the gel is denatured with alkaline solution (not heat as this would melt the gel) Denatured DNA transferred to a nitrocellulose or nylon membrane Radioactive/fluorescent probes used, complementary to the target sequence Hybridisiation occurs between probe and target DNA When unbound probe is washed away, and the membrane is exposed to photographic film, the target fragment will be visualised
72
What is the polymerase chain reaction?
PCR amplifies a specific DNA fragment from a small starting amount Requires two primers, complementary to each end of the target sequence Denature at 95℃ to separate strands Anneal at 55℃ so primers can bind to template DNA Extension at 72℃ so DNA polymerase can synthesise new strands Products appear as a single DNA band on a gel to help identify specific fragments compared to other gels Multiplex PCR uses multiple primer pairs to amplify several targets at once
73
Why is it more difficult to study RNA than DNA?
RNA is very unstable so requires very different isolation and storage techniques RNA is single stranded so you need formaldehyde during gel electrophoresis to denature the tertiary structure Often convenient to copy it into cDNA first using reverse transcriptase
74
What is Northern blotting?
RNA extracted from cells and denatured using formaldehyde Denatured RNA transferred to nitrocellulose membrane Probe (e.g. beta-globin gene) is added, complementary to target mRNA Washes remove any unbound probe Membrane exposed to film and developed Presence of beta-globin mRNA is detected
75
How can you make cDNA from RNA?
Start with single stranded mRNA with a 5' cap Add reverse transcriptase, oligo-T primer and dNTP pool The oligo-T primer recognises the strand, and allows snyhtesis of RNA:DNA hybrid from mRNA Then add DNA polymerase, the second primer, and another dNTP pool which allows the second strand to be synthesised Sequence of mRNA has been copied into a sequence of nucleotides that is identical to the cDNA copies
76
Describe the general organisation of prokaryotic chromosomes.
Prokaryotic genes arranged into operons (functional units including a group of genes transcribed together under the control of a single promoter)
Enables co-ordinated expression of genes that encode related proteins E.g. Lac operon in E.coli
77
Describe general organisation of eukaryotic chromosomes.
Condensed into chromatin, wrapped around histones Each chromosome has a centromere
Telomeres at the ends to prevent loss of genetic info 23 pairs in humans

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