Bacteriophage and Bacteriophage Lambda Flashcards
How many hydrogen bonds form between G-C and A-T?
G-C has 3, A-T has 2
Between which atoms do the hydrogen bonds of G and C form?
Of G: O6, N1, N2. At C: N4, N3, O2
Between which atoms do the hydrogen bonds of A and T form?
Of A: N6, N1. At T: O4, N3
What is the difference between a nucleotide and a nucleoside?
Nucleoside is base + sugar, nucleotide is base + sugar + phosphate
During DNA polymerisation, which groups join together (3’/5’ and functional group)
3’OH joins to 5’P
What is Chargaff’s rule?
The number of A/T and G/C is the same, therefore A must bond T and G bonds c
What is the difference between an endonuclease and an exonuclease?
Endonuclease cleaves DNA at multiple points internally, exonuclease cleaves sequentially (5’-3’) and more precisely
Where do restriction endonucleases originate from and what is important about their restriction sites?
Bacterial origin, palindromic recognition sites
Give an example of a restriction endonuclease which cuts to produce DNA with: 5’ overhang, 3’ overhand, no overhang
5’ - EcoR1, 3’-Pst1, none-Hpa1
What three things are required for ligation?
5’P, 3’OH and DNA ligase
What two types of ligation are there?
Sticky and blunt end
What is the mechanism of ligation (3 stages)?
- Adenylation of ligase (ATP/NAD addition causes AMP to bind to lysine)
- Activation of 5’P (AMP from ligase transferred to P in nick)
- Displacement of AMP (seals nick)
What does alkaline phosphatase do and what may it be used for?
Removes 5’P, can be used to prevent self-ligation (prevents ligation)
What is hyperchromicity?
The process of increase in A260 of DNA when it becomes single stranded
What is the role of topoisomerase type I and what is it’s method?
Uncoils DNA (nicks single strand, passes through loop and religates)
What is ethidium bromide used for and why?
Used as a fluorescent dye for DNA, it intercalates between DNA bases and fluoresces when exposed to UV light
What three types of bacteriophage are there?
Icosahedral w/ and w/out tails, filamentous
What are the 3 different types of genetic material a bacteriophage may have?
DNA/RNA, single/double stranded, circular/linear
What are the general life cycle stages of a bacteriophage?
- Initial phases - contacts cell, nucleic acid injected
- Early developmental - early genes expressed, nucleic acid replicated
- Late developmental - head/tail genes expressed, phage assembled
In bacteriophage, what does virulent and temperate mean?
Virulent: phage will always follow the lytic pathway
Temperate: phage may follow lytic or lysogenic pathway depending on conditions
What is a lysogen?
A bacteria cell with phage genome incorporated
What is a prophage?
Phage DNA incorporated into bacterial chromosome (occurs during lysogeny)
What is an induction event?
The process of excision of the phage genome from the host genome, the phage will follow the lytic cycle
In temperate phage what are the two things which will decide whether the phage undergoes the lytic or lysogenic pathway?
Conditions: If there are many nutrients/little competition for the cells then the phage will enter the lytic cycle
Multiplicity of infection: high MoI (viruses»>bacteria) causes lysogenic pathway to be entered
What method is used to determine whether phage are in lytic or lysogenic pathway and how?
Plaque assay: clear spots = lytic, turbid = lysogenic. Can determine whether phage are temperate or virulent also
What form does bacteriophage lambda undertake and what is the form of it’s genetic material?
Icosahedral head w/ tail, linear, double stranded DNA
How does the genetic material of bacteriophage lambda vary as it enters host cells?
The nucleic acid circularises (goes from linear to circular)
What are the 3 phases involved in the lytic cycle development of bacteriophage lambda and what occurs in these phases? (do not mention the genes/promoters)
Early phase: regulator genes expressed -> new RNA polymerase made, sigma factor and anti-termination factor
Delayed early phase: replication enzymes, 2nd regulator
Late phase: structural phage components, cell lysis stuff
What are the three phases involved in lytic cycle gene transcription (focus on promoters, genes, proteins produced and their effects)?
Immediate early: 2 promoter PR and PL are recognised by host cell transcriptional machinery and transcription of cro (PR) and N (PL) begins, tL1 and tR1 terminate transcription so only cro and pN are produced
Delayed early: pN anti-terminates (by acting on nut sites) causing transcription of Q, cII, cIII, 2xreplication, 5xrecombination and 2xDNA integrases
Late: initiates at PR’, pQ permits it to transcribe late genes (head & tail) lytic cycle begins
What genes in the control region are responsible for the lytic pathway or the lysogenic pathway?
Lytic: N, cro, Q
Lysogenic: cIII, N, cI, cII
What genes are present in the immunity region of bacteriophage lambda?
cIII –> cII (cIII, N, cI, cro, cII)
Why does superinfection of a host cell not usually occur when phage are in lysogenic state? How may superinfection occur?
If a new phage with the same lambda repressor as the original phage attempts to infect then the original lambda repressors would bind to the new phage’s OR/OL preventing it from tending lytic cycle
Describe the structure of the lambda repressor
27kDa, 2 domains (N & C), linker region –> dimer
What are the N and C terminals of the lambda repressors responsible for?
N: forms oligomers. C: binds operators
What promoters are responsible for cI expression?
PRM and PRE
How does the lambda repressor affect OL and OR?
OL: binds and prevents PL from initiating transcription of N
OR: binds and prevents PR from initiating transcription of cro, also stimulates PRM
How many alpha helices are present in the N-terminal domain of the lambda repressor and what are the roles of alpha 3 and 2?
5 alpha helices, 3 fits into major groove of DNA, 2 lies across major grove and H bonds to phosphate backbone
How many binding sites does each operator (OL/OR) have?
3
At regular lysogenic concentrations, which operator regions would have lambda repressor bound?
O1 and O2 (of both R and L)
How may the shape of the lambda repressor be described?
Dumbbell shaped
How does the binding of the lambda repressor to OR2 affect cI gene transcription?
When bound to OR2 the cI gene is transcribed due to interaction of lambda with PRM to enable more efficient binding of RNA polymerase
Give an example of a cooperative interaction that takes place involving OR/OL
When lambda repressor binds OR1, OR2, OL1, OL2 it forms an octamer between these, bending the DNA strand round, this stabilises the operators therefore a 3x lower lambda repressor concentration is required for O1, O2 binding
What happens if the lambda repressor concentration increases so that OR3 and OL3 are bound?
Binding to O3 would prevent expression of cI by PRM by preventing RNA pol binding
What are the roles of cII and cIII in lysogeny?
cII and cIII are responsible for the initial synthesis of the lambda repressor: cII binds to PRE allowing RNA polymerase to bind, cIII protects cII from degradation
How may a variation of cro transcription promote lysogeny?
cro may be transcribed the wrong way to create an antisense strand which may then bind to cro mRNA and inhibit expression
What 4 events are required for lysogeny?
- cIII & cII transcription (cII activates PRE to make lambda repressor)
- lambda binds to PR & PL & stops all gene transcription (PRE also stops as no cII/cIII)
- lambda binds to O1 & O2, activates PRM
- Before cII is switched off it turns on Pi/Panti-Q promoters which transcribe int/antisense Q, respectively
What is the role of cro and how does it achieve it’s role?
cro repressor prevents synthesis of lambda repressor by initially binding to OR3 (inactivating PRM), and then binding to OR1 & OR2, inactivating PR (and thus PRE - as no cII/cIII transcription)
