Nucleic Acids

Question 1

What does the term ‘genome’ refer to?

Answer 1

All of the genetic information of an organism.

Question 2

What does the term ‘nucleoside’ refer to?

Answer 2

Base and Sugar only.

Question 3

What is the key difference between purine and pyrimidine structures?

Answer 3

Purine bases are formed from a nitrogen rich heterocyclic double ring structure whereas pyrimidine base have only a single ring structure.

Question 4

Why is RNA less stable than DNA?

Answer 4

-OH group on Carbon-2 can act as a nucleophile to break phosphodiester link between nucleotides.

Question 5

What is the pitch angle of the DNA double helix and how long is each helical turn?

Answer 5

Pitch angle approx. 36degrees, each helical turn roughly 10.5 base pairs, 3.4 nm.

Question 6

Describe the minor and major groove.

Answer 6

• Major groove is is wide and shallow.
• Minor groove in narrow and deep.

Question 7

What is the persistence length?

Answer 7

The length of the DNA along which a thermally excited bend of 1 radian (~ 57°) typically occurs; a basic mechanical property quantifying the stiffness of a polymer.

Question 8

Why are short regions of DNA stiff?

Answer 8

• Electrostatic repulsion of phosphates.
• Compressive nitrogenous base stacking.

Question 9

Why and how does RNA form more complex structures than DNA?

Answer 9

• Majority of RNA exists as a single-stranded structure.
• It can, therefore, coils to form secondary structure by base pairing with other nucleotides.
• As there is no requirement for every nucleotide to form a base pair, this can lead to formation of hairpins, bulges and coils of more than 2 RNA strands.
• Formation of these complex structures allows RNA to perform additional structural and catalytic roles.

Question 10

What are the requirements for RNA Polymerase in E. Coli and what functions does it perform?

Answer 10

• Must unwind the DNA to form a transcription bubble - this exposes template strand.
• Does not require a free 3’ -OH like DNA polymerase, all 3 phosphates retained on first nucleotide.
• Catalyses synthesis of RNA from 5’ to 3’.
• Takes up free nucleotides to form the RNA.

Question 11

What are the 3 steps of the transcription cycle?

Answer 11

1. Initiation
2. Elongation
3. Termination

Question 12

What is the role of promoters in Transcription initiation?

Answer 12

• Required for initiation
• Defined by two sequences: Pribnow box (-10 bases relative to start of RNA transcription (+1)) and -35 region (clue is in the name!).
• Sigma factors bound to RNA polymerase recognise these promoter sequences. Different sigma factors recognise different promoters.

Question 13

How can promotors affect gene expression?

Answer 13

• Promoters have different strengths: stronger promoters have sequences closest to consensus sequence.
• Consensus sequences are the most optimal sequence for a specific sigma factors.
• Stronger promoters = increased gene expression compared to weaker promoters.

Question 14

What are the 2 transcription termination mechanisms?

Answer 14

• Rho-Dependent: Rho (enzyme) binds to ‘rut-site’ in RNA transcripts and uses energy from ATP hydrolysis to move along newly synthesised RNA, catching up with RNA Polymerase and pulling the RNA out of the RNA Polymerase enzyme.
• Rho-Independent: Relies on specific sequences in DNA called Dyad Symmetry sequences. Combination of hairpin AND run of Us required for termination.

Question 15

How can bacterial transcription be selectively inhibited?

Answer 15

• Rifampicin: Binds to β-subunit of RNA Polymerase preventing initiation but not elongation (no inhibitory effectswhen RNA is longer than 2-3 nucleotides).
• Actinomycin D: Intercalates into DNA preventing initiation AND elongation.

Question 16

What is the main type of DNA polymerase in E. Coli?

Answer 16

DNA Polymerase III

Question 17

What are the key features and requirements of DNA Polymerase III?

Answer 17

• Made up of 9 protein subunits.
• Synthesis does not depend on primary sequnce.
• Can only add nucleotides at the 3’ end of pre-existing DNA or RNA (must have free-OH).
• Requires Mg²⁺ to function.

Question 18

How is DNA replication initiated?

Answer 18

• Bi-directional replication forks generated by DNA helicase (process requires energy from ATP).
• Single -stranded binding proteins bind non-specifically to each strand, preventing reannealing.
• Primase creates short RNA primers, providing a free -OH for DNA polymerase to add nucleotides.

Question 19

How does DNA polymerase add nucleotides?

Answer 19

• Free 3’ OH- group nucleophilic attack to form phosphodiester bridge with phosphate nearest deoxyribose. Pyrophosphate (molecule of 2 phosphate ions) is released.
• Mg2+ and asp residues in catalytic site of DNA polymerase stabilises three phosphates.
• 3’ - 5’ exonuclease within DNA polymerase cleaves nucleotides one-at-a-time from the ends of the polynucleotides.

Question 20

How is DNA replication bi-directional and why it is discontinuous on the lagging strand?

Answer 20

• DNA Polymerase only works from 5’ to 3’
• Leading strand made continuously
• Lagging strand made discontinuously - requires many primers. Called Okazaki fragments.

Question 21

What is the role of DNA Polymerase I in DNA replication?

Answer 21

To remove RNA primers (it acts as a 5´-3´ exonuclease) and replace with DNA.

Question 22

What is the role of DNA ligase in DNA replication?

Answer 22

Catalysis of phosphate linkage between Okazaki fragments on lagging strand.

Question 23

List the 5 types of DNA repair in E. Coli.

Answer 23

Direct Repair
Mismatch Repair (MMR)
Base Excision Repair (BER)
Nucleotide Excision Repair (NER)
Homologous Recombination

Question 24

What is a synonym in terms of the genetic code?

Answer 24

Codons that code for the same amino acid, tend to have the same first two nucleotides, vary the 3rd.

Question 25

How has the genetic code evolved to minimise the effects of mutation?

Answer 25

• Mutations in 1st posisiton often result in similar amino acids.
• Mutations in the 3rd position are often silent.

Question 26

Why are insertion / deletion mutations more problematic than substitution mutations?

Answer 26

Frameshift - will affect all amino acids downstream.

Question 27

What is the role of amino acyl tRNA synthetases?

Answer 27

• Catalyse attachment of amino acid to correct tRNA molecule.
• Activates amino acids, providing energy to connect amino acid chain together.
• Contains editing pocket for proofreading: only incorrect amino acids fit into this site - they will be hydrolysed.

Question 28

How do repressors regulate transcription?

Answer 28

Protein factors that either bind to RNA polymerase or to operators that overlap with the RNA Polymerase binding site on the mRNA strand, both resulting in RNA polymerase being unable to bind to promoter, NO TRANSCRIPTION.

Question 29

How do activators regulate transcription?

Answer 29

Protein factors that bind to sites which do not overlap with the RNA Polymerase Binding Site. This helps RNA Polymerase to bind to the promoter sequence.

Question 30

What is an operon?

Answer 30

Unit of genomic DNA containing genes under the effect of a single promoter. Genes act together in a unified pathway.

Question 31

How does the Lac Repressor work in bacteria?

Answer 31

In the absence of the ligand (allolactose (a metabolite of lactose)), the Lac Repressor can bind to the operator sequence.
Lac Repressor cannot bind if lactose is present (allolactose will also be present).

Question 32

How does the positive control mechanism of the lac operon work in bacteria?

Answer 32

• When levels of glucose reduce, cAMP levels rise.
• cAMP binds to CAP, which binds to the operon, recruiting RNA polymerase.