Week 3 Flashcards
What is the difference between nucleoside and nucleotide?
- Nucleoside: base + sugar
- Nucleotide: base + sugar + phosphate
What direction is DNA replication in bacteria?
- Synthesised from 5’ to 3’ which forms phosphodiester bond (linked at 3’ hydroxyl).
What is the role of RNA primer in bacterial DNA replication?
- Provides free 3’ hydroxyl group that DNA polymerase can extend from.
What specific DNA polymerase is used in bacterial DNA replication and what does it do?
- Polymerase III holoenzyme has 3 core polymerase in each holoenzyme which binds to DNA and proof reads it.
- Its beta clap tethers core enzyme to DNA while clamp loader loads beta clamp.
- Can only add 5’ to 3’.
- DNA polymerase I is for lagging strand.
What is the role of replisome?
- Creates 2 replication forks.
What is a gene?
- a polynucleotide sequence that codes for a functional product.
What makes up a bacterial gene structure?
- promotor, leader, coding region, trailer and terminator
What role does the promotor region carry out?
- Recognition/binding site of RNA polymerase.
- It is not transcribed/translated
- 2 binding regions, one at -35 (recognition site) and another at -10 (binding site, Pribnow box)
What does the leader region contain and what is its function?
- Region between promoter and coding
- Transcribed but not translated
- Directs ribosome to bind here
- Contains transcription start site (shine-dalgarno sequence- important in initiation of translation)
Where is the coding region?
- Bounded by start and stop codon
- Transcribed and translated
Where is the trailer region and what is its role?
- Region after stop codon
- Transcribed and stops ribosome translation
What does the terminator region do?
- Signals RNA polymerase to stop transcription.
What is spontaneous mutation?
- From error in DNA replication
- Rare due to proof reading mechanisms
What is induced mutation and name a few examples?
- Exposure to mutagen
- Examples include base analogues, DNA-modifying agents, intercalating agents, UV radiation/ionising radiation
What are 4 types of mutations?
- Silent: no change
- Missense: changes codon
- Nonsense: change codon to STOP
- Frameshift: changes reading frame of gene
What does polycistronic mean in terms of RNA?
- Can encode for more than one gene
What is an operon?
- Genes that can be transcribed together
What does a sigma factor do?
- Separate protein part of enzyme complex that assist RNA polymerase to bind to particular promoters.
- Different sigma factors recognise different -10 and -35 promoters.
What are the 3 stages of transcription?
- Initiation, elongation and termination
What are some differences between prokaryotes and eukaryotes in terms of transcription?
- Prokaryotes have 1 type of RNA polymerase whilst eukaryotes have 3.
- Eukaryotes have 5’ caps on mRNA and poly A tail whilst prokaryotes have none.
- Prokaryotes have sigma factors whilst eukaryotes have several transcription factors.
- Eukaryotes have exons and introns whilst prokaryotes don’t.
What are 2 ways genes are regulated?
- Regulation of gene expression (transcription initiation, elongation, translation)
- Alter activity of enzymes and proteins (post-translational)
What are inducible genes?
- Expression (transcription) turned on by stimulus. eg.metabolism
- B-galactosidase is an inducible protein
What are repressible genes?
- Expression (transcription) turned off by stimulus. eg. bio synthetic pathway
- lac operon or trp operon
What regulation mechanisms are used at level of translation?
- Regulation by riboswitch where mRNA directly binds to metabolite causing it to fold differently (impedes access to ribosome binding sites)
- Small RNA regulation where antisense RNAs bind to mRNA leader sequence and inhibit ribosome attachment and translation.
What are some mechanisms used for global regulation?
- regulatory proteins
- alternative sigma factors
- two component signal transduction system where 2 proteins govern the pathway. (sensor kinase: extracellular recpetor for metabolite, response-regulator protein: activated by sensor kinase and can enhance transcription (activator) or inhibit transcription (repressor).
Describe the structure of plasmids.
- usually double stranded circular DNA (some are linear)
- usually supercoiled by DNA gyrase
- can have more than one type of plasmid per cell
What is the function of plasmid?
- Contain non-essential genes for bacterial growth
What is the function of plasmid in B.anthracis?
Produce toxins for mammalian cells
What is the function of plasmid in B.thuringiensis?
Produce toxin used in GM crops
What is the function of plasmid in B.cereus?
Produce toxins that cause food poisoning
What are 3 types of plasmid replication?
- Theta replication: unidirectional and bidirectional
- Rolling circle replication
What is theta replication?
- Most common plasmid replication.
- Same mechanism as chromosomal replication
What is rolling circle replication?
- When one strand is nicked, the 3’ end serves as primer and DNA polymerase synthesise complementary strand from here.
- Intact strands serve as template and displaces original second strand. Displaced ssDNA is then converted to dsDNA circle.
What is host range? Name the two types.
- The species of bacteria in which the plasmid can replicate.
- Broad host range replicate in many species (often encode own replication proteins).
- Narrow host range restricted to single species (often rely on host proteins for replication).
What is the copy number?
- Avg number of a single plasmid tpye per cell
What do incompatibility group mean?
- Plasmids with similar mechanisms of replication are incompatible.
What is a transconjugant?
- recipient cell that has received DNA as a result of conjugation.
Describe the mechanism for DNA transfer in bacteria.
- Donor cell produce pilus (encoded by plasmid tra genes)
- Pilus contacts potential recipient
- Retraction of pilus brings cells into close contact and a pore forms in adjoining cell membranes
- In rolling cricle replication, new complementary DNA strand is synthesised in the donor
When is bacteria considered transformed?
- When uptake of naked DNA is successful (transformant).
- Bacteria that are able to do this are called competent.
What does PCR require?
- primers, DNA polymerase, dNTPs, template strand, some co-factors
What is a primer and describe 2 types?
- short DNA or RNA strands that primes DNA synthesis
- can incorporate restriction enzyme sites
- PCR requires both forward (analogous to target sequence) and backward (complementary to target sequence) primers.
