What does the term ‘genome’ refer to?
All of the genetic information of an organism.
What does the term ‘nucleoside’ refer to?
Base and Sugar only.
What is the key difference between purine and pyrimidine structures?
Purine bases are formed from a nitrogen rich heterocyclic double ring structure whereas pyrimidine base have only a single ring structure.
Why is RNA less stable than DNA?
-OH group on Carbon-2 can act as a nucleophile to break phosphodiester link between nucleotides.
What is the pitch angle of the DNA double helix and how long is each helical turn?
Pitch angle approx. 36degrees, each helical turn roughly 10.5 base pairs, 3.4 nm.
Describe the minor and major groove.
- Major groove is is wide and shallow.
- Minor groove in narrow and deep.
What is the persistence length?
Perameter used to quantify the stiffness of a molecule (in this case DNA).
Why are short regions of DNA stiff?
- Electrostatic repulsion of phosphates.
- Compressive nitrogenous base stacking.
Why and how does RNA form more complex structures than DNA?
- 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.
What are the requirements for RNA Polymerase in E. Coli and what functions does it perform?
- 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.
What are the 3 steps of the transcription cycle?
- Initiation
- Elongation
- Termination
which 2 sequences?
What is the role of promoters in Transcription initiation?
- 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.
How can promotors affect gene expression?
- 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.
What are the 2 transcription termination mechanisms?
- 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.
How can bacterial transcription be selectively inhibited?
- 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.
What is the main type of DNA polymerase in E. Coli?
DNA Polymerase III
What are the key features and requirements of DNA Polymerase III?
- 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.
How is DNA replication initiated?
- 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.
Mechanism, stabilisation and proofreading.
How does DNA polymerase add nucleotides?
- Free 3’ OH- group on the RNA primer acts as a nucleophile and attacks nearest P to form phosphodiester bridge. Pyrophosphate (molecule of 2 phosphate ions) is released.
- Mg2+ and asp residues in catalytic site of DNA polymerase stabilises three phosphates of new incoming nucleotide..
- 3’ - 5’ exonuclease within DNA polymerase cleaves any INCORRECT nucleotides.
How is DNA replication bi-directional and why it is discontinuous on the lagging strand?
- DNA Polymerase only works from 5’ to 3’
- Leading strand made continuously
- Lagging strand made discontinuously - requires many primers. Called Okazaki fragments.
What is the role of DNA Polymerase I in DNA replication?
To remove RNA primers (it acts as a 5´-3´ exonuclease) and replace with DNA.
What is the role of DNA ligase in DNA replication?
Catalysis of phosphate linkage between Okazaki fragments on lagging strand.
List the 5 types of DNA repair in E. Coli.
Direct Repair
Mismatch Repair (MMR)
Base Excision Repair (BER)
Nucleotide Excision Repair (NER)
Homologous Recombination
What is a synonym in terms of the genetic code?
Codons that code for the same amino acid, tend to have the same first two nucleotides, vary the 3rd.
