APS138 Cell And Molecular Biology - Ton Flashcards
Prokaryotic genes are often organised in…
Functional operons
What is the operator region?
A segment of DNA that the repressor or activator protein binds to
2 parts to a functional operon:
Promoter and functional genes
Activator proteins (which activate RNA polymerase) coded by…
Activator genes
How can the activator protein stability affect the expression of a gene?
If activator protein unstable then it is degraded more easily and won’t activate RNA polymerase as much, so the gene won’t be expressed as much
What is the lac operon found in?
E. coli gram negative bacteria
What is the lac operon expression regulated by?
Repressor protein coded for by Lac I repressor gene
What does lactose bind to?
Lac I repressor protein - inactivates and no longer fits operator, operon is expressed - lactose can be broken down - negative feedback regulation
- b-galactosidase is the enzyme
What does Trp stand for?
Tryptophan - an amino acid
What does the trpR repressor gene code for?
An inactive (by default) repressor protein - tprR - activated by tryptophan - binds to operator and blocks expression of tryptophan - negative feedback
Explain quorum sensing in bacteria
When cell density is low bacteria display individual behaviour. They constantly release low levels of auto-inducer compounds (often homoserine lactone), which naturally diffuse when bacteria are at low levels. However, when cell density is higher, group behaviours are displayed, such as virulence, microfilm formation or luminescence, as the concentration of the auto-inducer compounds are higher and diffuse back into cells, triggering reactions and gene expression.
- Hawaiian bobtail squid lives in symbiosis with bioluminescent bacteria (Aliivibrio fescheri) - lux operon - luxR repressor activated by enzyme Lux I which is constantly secreted at a low rate.
What is a nucleosome?
8 histones + 146 base pairs of DNA
What is heterochromatin?
Dense, tightly packed chromatin - inaccessible for transcription
What is lightly packed chromatin called?
Euchromatin
How is heterochromatin converted into euchromatin?
Histone acetyl transferases and histone methyl transferases
acetylation
How is euchromatin converted into heterochromatin?
Histone deacetylase and histone demethylase
methylation
In the paused state of RNA polymerase II what is phosporylated?
Serine 5
In the active state of RNA polymerase II what is phosphorylated?
Serines 2 and 5
Where does alternative splicing occur?
In the nucleus
What are the two types of small RNAs?
microRNA (miRNA) and small interfering RNA (siRNA)
What are small RNAs?
Double stranded RNA between 20 and 30 nucleotides long - can regulate heterochromatin methylation
What is microRNA and siRNA used for
Repressing gene expression via three mechanisms:
1. repression of gene translation
2. Promoting mRNA degradation
(post-transcriptional silencing)
3. Repression of gene transcription via chromatin remodelling and DNA methylation (siRNA) - thought to be ancient way of protecting plants from retroviruses
What enzymes cut pre-miRNA into miRNA?
Dicers
What degrades unneeded proteins?
Proteasome
Breaks them down in to small protein fragments (peptides)
What are proteins tagged with before degradation?
Multiple Ubiquitins
Miss out Figure 5 of the paper
Zooweemama
What does reverse-transcriptase quantitative PCR (RT-qPCR) measure?
Relative mRNA levels in tissues/cells
What are the 3 stages of PCR? What temperatures are required
- Denature at 95 degrees
- Anneal gene-specific primers are 55 degrees
- Extend primers using taq DNA polymerase at 72 degrees
Repeat cycle 35 times
What do nuclear run-on assays measure?
Rates of mRNA production from the nucleus
How do you carry out a run on assay?
- Rapidly extract nuclei
- Add biotin-U and unlabelled A, G and C
- Extract biotin-labelled mRNA (mRNA which has been produced since extracting the nucleus) using streptvidin-coated beads
- Measure biotin labelled mRNA by RT-qPCR
What is ChIP?
Chromatin-immuno-precipitation. Used to quantify binding between specific gene DNA sequences and a protein - measures the amount of any specific DNA sequence attached to a protein
How is ChIP carried out?
Chromatin extracted from nuclei and sonicated to chop into chunks. Immunoprecipitated using a specific antibody against the protein. Extract DNA from chromatin. qPCR to determine levels of DNA in chromatin before and after immunoprecipitation.
What is H3K4me3 a good marker for?
Open euchromatin
