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Foundation Of Health and Medical Practice > Molecular Biology (DNA, RNA, Protein synthesis, sequencing) > Flashcards

Molecular Biology (DNA, RNA, Protein synthesis, sequencing) Flashcards

1
Q

What does a mediator complex do? (To do with transcription in eukaryotes)

A

Acts as a central switchboard which integrates multiple inputs from the regulated expression of the genome.

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2
Q

Which bases are Purines?

A

A, G

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3
Q

Which bases are Pyrimidines?

A

C, T, U

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4
Q

What does a nucleoside consist of?

A

A nitrogenous base and pentose sugar

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5
Q

What does a nucleotide consist of?

A

A nitrogenous base, pentose sugar and triphosphate group

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6
Q

Name each base as a nucleoside

A

Adenosine, Thymidine, Cytidine, Guanosine, Uridine

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7
Q

What is the difference between ribose and deoxyribose structure?

A

Ribose has a hydroxyl group on the 2’ carbon, deoxyribose has just a H

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8
Q

What are the names of the free nucleotides used in semi conservative replication?

A

dATP, dGTP, dTTP, dCTP - these are all dNTPs.

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9
Q

What are the requirements for semi conservative replication?

A

1) Requires a template strand - mother strand of DNA
2) DNA polymerases can only function in a 5’ to 3’ direction to form phosphodiester bonds
3) Requires dNTPs as substrates
4) Requires a primer - short sequence of 10 RNA nucleotides. Because DNA polymerase needs a free 3’ hydroxyl to add nucleotides to

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10
Q

Describe the first stage of DNA replication

A

Strand separation. Dna A protein binds to strand and begins to separate. Dna B protein is a helicase so unzips strand further (using energy from ATP). Then SSB single stranded binding protein prevents reannealing (spontaneous rebinding of the strands).

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11
Q

Describe the second stage of DNA replication

A

Primase attaches RNA primers to the strands to initiate DNA synthesis. The leading strand is 5’ to 3’ towards the replication fork, and this is synthesised continuously by DNA polymerase. The lagging strand that runs in the opposite 5’ to 3’ direction AWAY from replication fork needs multiple RNA primers as this is synthesises in smaller segments away from the replication fork.(opposite direction as DNA polymerase can only run in 5’ to 3’ direction.)

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12
Q

What are the smaller segments synthesised on the lagging strand called?

A

Ozaki fragments

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13
Q

What is the meaning of exonuclease?

A

Enzyme that cleaves (cuts) nucleotides from the end of a polynucleotide chain.

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14
Q

Apart from synthesising DNA, what is another function of DNA polymerase?

A

DNA polymerase also has a exonuclease action to remove the RNA primer from the lagging strand. This is because DNA polymerase will run into RNA primers, so to avoid the DNA polymerase removes RNA primer to leave a gap. DNA ligase can then form the new phosphodiester bonds of the Osaka fragments.

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15
Q

Apart from removing RNA primers, what is another exonuclease action of DNA polymerase?

A

In the 3’ to 5’ direction DNA polymerase can proofread the DNA strand that is being formed.

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16
Q

How does DNA polymerase proofread the new DNA strand?

A

If a wrong nucleotide is synthesised the active site of the DNA polymerase is distorted. This activates the 3’ to 5’ activity to cleave the incorrect nucleotide. DNA polymerase can the continue the correct base pairing.

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17
Q

What is the difference between dNTPs and ddNTPs?

A

ddNTPs do not have a hydroxyl OH group on the 3’ carbon on the ring. Full name is dideoxynucleotides.

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18
Q

Outline the process of Sanger sequencing (1)

A

Firstly the DNA being sequenced has to be amplified (multiple copies made). Heat is then used to denature the DNA so the strands separate. Then an RNA primer is joined to the 5’ end of the template strand. This “primed” DNA is separated into 4 reaction vessels. Then DNA polymerase and all 4 dNTPs are added to the reaction vessels.

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19
Q

Outline the process of Sanger sequencing (2)

A

Then specially modified ddNTPs are added, one to each reaction vessel. In the vessel the DNA polymerase attached the dNTPs at the primer until the ddNTP is paired. Here sequence is terminated as ddNTPs do not have a OH on the 3’ carbon. Results in DNA fragments of different lengths.

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20
Q

Outline the process of Sanger sequencing (3)

A

Polyacrylamide gel electrophoresis is used to sequence the DNA. When power supply is turned on DNA migrates from the negative pole to the positive pole (because phosphate backbone of DNA has a negative charge). Smaller lighter lengths migrate to bottom of gel, forms band patterns. Gel is precise, can separate strand that differ by only one pair. Sequence read from bottom of plate upwards. Results in complementary sequence of DNA sample being found.

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21
Q

Outline the process of NGS (next generation sequencing)

A

Firstly, large DNA molecules are ligated (fragmented) into short adaptors. This is then attached to a surface in a chamber. PCR can be used to amplify the number of a particular strand of DNA. This immobilises the DNA. Then all 4 dNTPs are added, and these have a fluorescent dye and a terminator (which can cleave the strand) attached to it. One cycle of this process corresponds to one nucleotide being added to the sequence, therefore one colour being generated, as the terminator cleaves the strand. The fluorescent dye allows to see the complementary strand to the DNA strand that was placed on the plate initially, therefore the DNA strand can be sequenced.

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22
Q

Outline the process of PCR

A

PCR allows a specific region of the gene to be amplified. Firstly the DNA strand is denatured with high temperatures around 72-95 degrees. This separates the DNA strands. Then specific primers that are complementary to the start and end of the gene are added. These anneal (bind) to the DNA strand when the sample is cooled to around 50 degrees.
Final stage is addition of Taq DNA polymerase (this is heat stable). This extends the piece of DNA. The cycle can then be repeated, the product of the first cycle becomes the template of the next cycle. This allows many copies of one segment of DNA to be produced, useful for further manipulation of DNA.

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23
Q

Which DNA sequencing process is exponential?

A

PCR is exponential, meaning that 2 cycles = double the number of DNA strands produced. Exponential amplification.

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24
Q

Why is RNA considered the “centrepiece” of life?

A

DNA used for storing information, proteins carry out structural and functional roles, however RNA can carry out all these roles during transcription (RNA polymerase) and translation (mRNA, tRNA, rRNA).

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25
Q

If RNA can carry out all roles, why did DNA and protein develop?

A

DNA is more stable than RNA for information storage. Proteins are better in a catalytic role as there are 20 different amino acids so more levels of structure so proteins can have a much higher level of specificity.

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26
Q

What is the difference in RNA polymerase between the domains of life?

A

Bacteria RNA pol has 4 subunits, Archaea RNA pol has 12 subunits, Eukaryotic RNA pol has 12-17 subunits. The functionality (suits the purpose)of RNA pol increases from Bacteria to Eukaryotes.

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27
Q

What are the 2 roles of RNA polymerase?

A

RNA polymerase has roles in polymerisation (production of mRNA), and in nucleolysis (cleaving mistakes in the strands).

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28
Q

Which part of DNA/RNA polymerase prevents the enzyme from dissociating from the template strand?

A

The DNA clamp is a protein complex that binds the enzyme to the template strand.

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29
Q

What is the first stage of transcription?

A

Initiation: Transcription factors bring RNA pol to the promoter, where it binds to the 5’ end. (RNA pol works in the 5’ to 3’ direction.)

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30
Q

What is the second stage of transcription?

A

Elongation: Transcription factors and rNTPs that are present allow mRNA to be synthesised along the antiparallel template strand. The mRNA strand is the same as the non-template strand of DNA (but U replaces T).

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31
Q

What is the third stage of transcription?

A

The RNA pol reaches a stop signal/codon. Causes the RNA pol to detach then restart in order to make multiple transcripts of the same gene.

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32
Q

What happens to pre mRNA before the strand can diffuse to the cytoplasm?

A

mRNA strand is spliced, introns are removed to produce mature mRNA.

33
Q

What is an operon?

A

A functioning unit of DNA containing a cluster of genes under the control of a single promoter.

34
Q

What is the difference between prokaryotic operons and eukaryotic operons?

A

Prokaryotic operons generally produce polycistronic mRNA (single mRNA codes for several proteins) whereas eukaryotic operons code for monocistronic mRNA (single mRNA codes for a single specific protein).

35
Q

What is the difference between transcription in prokaryotes and eukaryotes?

A

In prokaryotes transcription and translation are carried out simultaneously due to lack of nucleus. In eukaryotes there are internal membranes therefore the processes are separated.

36
Q

What is a promoter?

A

Promoters are the DNA sequence where RNA pol binds. They are upstream of the structural gene start site. Bacterial promoters contain 2 hexanucleotide sequences are -35 and -10.

37
Q

What is an operator?

A

A region within the operon where the repressor binds to.

38
Q

What is a repressor?

A

Any protein that binds to DNA and regulates the expression of genes by decreasing the rate of transcription.

39
Q

How do enhancers up regulate (positive) gene expression?

A

Enhancers bind transcription factors to recruit RNA polymerase. This therefore increases the frequency of the transcription initiation complex forming = increased expression of that gene.
Doesn’t have to be close to the gene to have an effect.

40
Q

What do silencers do?

A

Silencers down regulate (negative) gene expression. Doesn’t have to be close to the gene to have an effect.

41
Q

How is the breakdown of lactose regulated by the lac operon?

A

The lac operon is inducible, therefore is usually repressed unless in the presence of lactose. The lac operon controls transcription of the gene that produces beta galactosidase (which breaks down lactose). When lactose is present it binds to the repressor so the repressor cannot block RNA pol from transcribing the gene. Means the enzyme beta galactosidase is not produced unless the substrate is present.

42
Q

What form the transcription initiation complex?

A

RNA polymerase, TFIIF, TFIIE, TFIIH

43
Q

How do transcription factors, the initiation complex and RNA polymerase interact?

A

Genes loop to force transcription factors to interact with each other and RNA polymerase. One RNA pol can form an interface (common boundary) between up to 2000 transcriptional factors - this means mediators are required to allow interaction between transcription factors and the initiation complex.

44
Q

Outline the process of splicing mRNA.

A

First the 5’ cap (called 7-methylguanosine) is added as this is needed in translation initiation. Splicing converts the freshly generated mRNA (premRNA) into mature mRNA. This is catalysed by the spliceosomes (snRNPs) that remove introns. A Poly A tail is added to the 3’ end for stability of the mRNA and prevents degradation.

45
Q

How does gene regulation give rise to a wide diversity of proteins?

A

Transcription regulation, epigenetics, mRNA splice variants, mRNA editing.

46
Q

Apart from transcription factors and operons, what else can control the transcription of specific genes?

A

Hormones and Drugs

47
Q

How can drugs affect transcription?

A

Drugs may target nuclear/plasma membrane receptors, which can directly alter DNA, or cause signalling cascades which may activate or repress transcription factors (therefore either up or down regulates expression of a gene

48
Q

How can hormones affect transcription?

A

Steroid hormones may interact with nuclear membrane receptors to alter gene expression(similar to drugs). Protein hormones may bind to plasma membrane receptors and cause a signalling cascade.

49
Q

What kind of mutation causes b thalassemia due to insufficient b-globin?

A

Splicing mutations

50
Q

What kind of mutation involves swapping one nucleotide for another to form a different codon?

A

Mis-sense mutations

51
Q

What is a nonsense mutation?

A

Swapping a nucleotide to form a stop codon

52
Q

What is a silent mutation?

A

Swapping a nucleotide to form the same codon (will still have less effective translation as the tRNA molecule will still be different.)

53
Q

What can cause frame shift mutations?

A

Insertion and deletions of codons, as it results in the code not being in multiples of three.

54
Q

What is alternative splicing?

A

When different combinations of splice sites on mRNA are chosen. This produces variably spliced mRNA (responsible for antibody variability).

55
Q

What is the result of increase DNA methylation on a gene promoter?

A

This silences that particular gene and it doesn’t allow transcription factors to access the DNA, therefore results in transcriptional inactivation.

56
Q

Describe the structure of a tRNA molecule

A

Has an anticodon that is complementary to the codons on mRNA in the opposite 5’ to 3’ direction. It also has an attachment for the amino acid it has a code for at the top. It binds an amino acid to the acceptor arm.

57
Q

What is required for an amino acid to attach to a tRNA molecule?

A

Amino acid, an enzyme specific for that tRNA+amino acid and ATP(energy)

58
Q

Which type of ribosome has a 50S large subunit (34 proteins, 5S rRNA, 23S rRNA) and a 30S small subunit (21 proteins and 16S rRNA)?

A

Bacterial ribosomes

59
Q

Which type of ribosome has a 60S large subunit(49 proteins, 5S rRNA, 5.8S rRNA, 23S rRNA) and a 40S small subunit (33 proteins and 18S rRNA)?

A

Eukaryotic ribosomes

60
Q

Which subunit is responsible for decoding genetic information and ensuring correct translation?

A

The smaller subunit

61
Q

What is the large subunit of a ribosome responsible for?

A

Involved in peptide bond formation and peptide transferase activity (connecting amino acids together in the polypepetide). It also has ribosomal exit tunnels where the polypeptide begins to get folded as it comes out of the ribosome.

62
Q

How is protein charge distributed in a ribosome?

A

Negative charge in centre (due to phosphodiester backbones) and positive charge surrounding this to neutralise the charge.

63
Q

How does the ribosome attach to mRNA in prokaryotes?

A

The sequence on the mRNA ribosome binding site is complementary to the 16S rRNA region (Shine-Dalgarno)

64
Q

Why in eukaryotes does one mRNA correspond to one gene?

A

Because eukaryotes do not have multicistronic mRNA or operons therefore one mRNA is recognised by a ribosome once.

65
Q

How does the ribosome attach to mRNA in eukaryotes?

A

There are Kozak sequences on mRNA that are recognised by leading regions of the mRNA strand. (it is not facilitated by direct base pairing between rRNA and mRNA)

66
Q

What are the 3 regions of a ribosome?

A

There is an aminoacyl tRNA site (A), peptide tRNA (P) and an exit (E) site.

67
Q

What is the role of initiation factors in translation?

A

They are needed for initiation and include a GTPase.

68
Q

Which factor in translation has 2 GTPases?

A

Elongation factors needed for elongation. The energy released by hydrolysis of GTP causes conformational changes.

69
Q

Which factor in translation is needed for termination?

A

Termination/Release factors are needed, and they include one GTPase and one ribosome recycling factor.

70
Q

Outline the process of initiation in translation

A

First the small ribosome subunit binds to the Kozak or Shine-Dalgarno sequence and forms a 30/40S initiation complex at the start codon. (mRNA + small subunit + methionine tRNA).
GTP is then hydrolysed to GDP and Pi(inorganic phosphate), and the energy released removes the initiation factors. This is a 70/80S initiation complex (mRNA, small+large subunit, methionine tRNA)

71
Q

Outline the process of elongation in translation (bacteria)

A

When the tRNA binds to the amino acyl (A) site it binds reversibly as a complex with EF-Tu GTP. This is an elongation factor that catalyses the amino acyl tRNA to bind to a ribosome. The anticodon of the tRNA can interact with the mRNA codon on the A site, the end bound to the amino acid is also bound to EF Tu. GTPase is then activated to hydrolyse GTP. The GDP produced that is bound to the EF-Tu then dissociates from the ribosome. The peptide transferase reaction then occurs (formation of peptide bond), where GTP is hydrolysed to shift the mRNA to 3’ and translocate the peptide tRNA from the A site to P site. The deacylated tRNA shifts to the E site. The growing chain is added to the A site and translocated to the P site until the next amino acid is added.

72
Q

What happens if the wrong tRNA attaches to the A site in translation?

A

The tRNA is ejected from the complex and the whole process is repeated until the correct tRNA is bound to the mRNA codon.

73
Q

Outline the process of termination in translation (bacteria)

A

The stop codon on mRNA causes a release factor to bind and promote hydrolysis between peptidyl-tRNA (P) and the final amino acid. The polypeptide is released and the complex dissociates by hydrolysing GTP.

74
Q

What is an amino acid tRNA synthesise?

A

An enzyme that catalyses the addition of an amino acid to a tRNA. This enzyme is specific for each tRNA-amino acid combination. Hydrolysis of ATP is needed for this reaction. Results in a loaded tRNA that can then be used in translation.

75
Q

What is the wobble hypothesis?

A

There are 61 amino acid codons (64 but 3 are stop codons) but only 40 tRNA codons. Means that multiple codons can bind to the same tRNA. This happens as the 3rd base in the codon can pair with more than one base pair - A on the codon can bind with either G or U. This corresponds to the first base on the anticodon of the tRNA. This is therefore called the wobble position.

76
Q

Name 3 antibiotics that interfere with translation in order to fight bacterial infection

A 
Tetracycline = Inhibits aminoacyl-tRNA binding from binding to the A site so that particular mRNA strand will not be translated.
Erythromycin = binds to P site(peptide transferase) so blocks polypeptide from exiting ribosome, prevents translation.
Chloramphenicol = blocks correct positioning of the aminoacyl-tRNA for peptide transferase reactions, so translation cannot occur.
77
Q

Name 2 toxins that interfere with translation to prevent ribosomal function

A

Ricin = targets eukaryotic and prokaryotic cells, prevents activation of translation factor GTPases (no translation without hydrolysis of GTP)
Diptheria toxin = acts on eukaryotic elongation factor, binds an ADP-ribose to the factor which inactivates it, so elongation in translation cannot occur.

78
Q

How does the ratio of RNA and protein change in different evolutionary stages?

A

There will be a higher protein content in faster evolving systems, and a higher RNA content in more primitive systems.
This is because proteins are more versatile and specific as catalysts than RNA.

Foundation Of Health and Medical Practice (8 decks)

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