week 1 Flashcards
How is DNA organised to fit into a prokaryotic cell?
Chromosome is compact due to DNA supercoiling
Is DNA usually supercoiled positively or negatively? and why?
negatively to favour unwinding as local unwinding is needed for DNA recombination
How is DNA organised in eukaryotes?
packaged tightly into chromatin
How is chromatin stabilised?
stabilised by nucleosomes (DNA wrapped around histone)
What is the definition of euchromatin and heterochromatin?
- least condensed (rich in genes)
- most condensed (very few genes)
Many genes are arranged in clusters controlled by the same ___
promotor
Explain lac operon when lactose is present vs absent
- when lactose is present, it binds to lac repressor protein and distorts its shape so it can no longer bind to operator region. RNA polymerase binds to promotor region and transcription of structural genes take place, producing lactase in the presense of lactose
- lactose is not present, the shape of the repressor protein is not distorted, therefore it binds onto the operator region blocking the RNA polymerase. As a result, the gene is switched off and no lactase is made
What are plasmids?
pieces of DNA that contains non-essential genes, beneficial but not necessary
In eukaryotes, each gene has their own p_____.
promotor
eukaryotic genes contain exons and introns. what are they?
exons = coding regions
introns = non-coding regions
What is the benefit of having introns?
alternative splicing of mRNA = different protein structures
Describe the chromosomes in prokaryotes
- circular chromosome
- not membrane bond
- nucleiod = region of DNA with associated proteins
Describe the chromosomes in eukaryotes
- linear chromosomes
- in pairs (homologues)
- 2 copies of each gene in each homologous chromosome
Why are chromosomes more visible in prophase?
condensing of chromatin
What is the chromosome territory?
chromosomes are arranged in a defined area of the nucleus.
Compare sizes and density of genome prokaryote vs eukaryote
pro:
- small genome
- genes are close to each other
- high gene density
eu:
- large genome
- low gene density
- only 1% codes for proteins
What are non-coding functional elements identified as?
- TF, microRNAs, interferring RNAs, long non coding RNAs
What is syntheny?
comparing the organisation of human genome with other species can elucidate how species evolutionarily diverged. The genome of 2 species can be arranged into blocks where the order of genes is the same a their most recent ancestor.
Why do most prokaryotic genes not contain introns?
- introns would inhibit translation coupled to transcription in prokaryotes
- introns appeared after eukaryotes diverged from prokaryotes
What is the central dogma?
genetic can be transferred between RNA & DNA (transcription and reverse transcription) and to Protein (translation) but it can not be transferred from protein back to nucleic acids.
Explain transcription
RNA polymerase synthesise RNA strand using DNA as a copy strand
In bacteria, transcription and translation are ___
coupled
Why is transcription/translation coupled in bacteria?
usually in eukaryotes transcription takes place in the nucleus and translation takes place in ribosomes in cytoplasm. however bacterias don’t have nucleus so ribosomes can translate whilst its still being transcribed
Gene has x-axis: what is +1
First nucleotide of the transcript
Gene has x-axis: what is -10 and -35
-10 = TATA box
- they are important for RNA polymerase to recognise the promotor so they know where to bind and start transcribing
- monocistronic mRNA: ____
- polycistronic (multigenic) ____
- one coding sequence = 1 protein
- operons (one gene multiple proteins)
prokaryotes have a single RNA polymerase core for all genes. explain the structure.
consists of 5 subunits, sigma factor has to join for active transcription. sigma factor required to recognise -10 and -35. sigma leaves when transcription starts bc it is no longer needed
how does RNA polymerase know its in the transcriptional terminator?
short hairpin in the region of termination followed by a stretch of Us in the transcript. because the AU pair is weaker than CG, it causes the pairs to dissociate.
There is a ____ equilibrium between occupied and unoccupied state of a promotor
dynamic
Describe the equilibrium in a strong promoter setting and weak promotor setting.
equilibrium shifts towards the bound molecule. so more transcripts are made.
in the case of a weak promotor situation, equilibirum shifts towards the promotor free situation. so less transcripts are made and less of the subsequent proteins are created.
What are the 2 ways to compare promotor strength?
- GFP
- enzymatic reporter gene
How to use GFP to compare reporter strength?
using fluorescent reporter gene coding for GFP (green fluorescent protein). if we fuse promotor 1 an d2 to GFP and look at cells, we will see that the brighter and fluorescent, the stronger the promotor. because stronger promotor → more GFP mRNA is made → more fluorescence protein → brighter colour
How to use enzymatic reporter gene to compare reporter strength?
lacZ codes for B-galactosidase (enzyme) which catalyses the reaction where a di-sugar (no colour) is broken down into 2 mono-sugars (one being yellow). the more intense the reaction, the more yellow the colour = stronger promotor
4 ways to change the amount of protein produced (altering gene expression)
- transcriptional level = more RNA transcripts → more proteins
- post-transcriptional level = more stable RNA transcripts → more proteins
- translational level = premature translation termination → less protein
- post-translational level = protein degration → less protein; activation or inactivation of a protein by covalently attatching phosphate, acetyl, sumo, ubiquitin and other groups (characteristics of eukaryote not prokaryotes
How does alternative sigma factors activate specific sets of genes?
sigma factor tells the core enzyme which promotors to bind to in order to express certain genes
The -10 and -35 boxes are recognised by sigmas and they have different sequences for different sigma factors.
What is a regulon?
the genes that are triggered by different sigma factors are scattered throughout genome but are all grouped in regulon.
sigma independent regulation using transcription factors. divided into 2 groups
repressors or activators
Describe negative gene regulation using repressors
- transcription by default is on, if theres no transcription factor than transcription will take place
- if transcription factor is in place and it codes for a repressor protein, then repressor can shut down transcription by binding at the promotor, physically blocking RNA polymerase from binding to promotor
- repressor binds to operator region
Describe positive gene regulation using activators
- RNA polymerase has a weak binding ability to promotor (hard to bind due to -10 and -35 regions that binds to sigma)
- if an activator is in place, it binds on the promotor region (activator binding site)
- it undergoes a reaction where the activator anchors the RNA polymerase at the promotor, thereby helping transcription
Lac operon is weak even with repressor inactive so we require an activator called CAP. CAP can’t bind unless _____ is present
____ is a regulatory molecule anti is ____ by glucose
if glucose isn’t present ____ is made and binds to CAP and CAP is bound to ____
cAMP
cAMP
inhibited
cAMP
activator binding site
Both CAP and Lack is regulated by cAMP and lactose through conformational change of proteins. these effectors bind to ______ so this type of regulation is called ____
allosteric site
allosteric regulation
What is lactose made of?
glucose and galactose
What causes lactose intolerance>
decreasing expression of the LCT gene. leads to sustained lactase production
Describe the 3 levels of expression of the lactase gene
- lac repressor bound
- lac repressor not bound but activator not bound
- lac repressor not bound activator anchors RNA polymerase
What are transposons?
mobile elements which can jump from one DNA molecule to another (e.g. plasmid to chromosome), codes for antibiotic resistance genes
What is horizontal gene transfer?
using transposons to migrate between bacteria and spread antibiotic resistance
What are smaller plasmids used to construct?
clowning vectors used carry DNA fragments of interest
Describe bacterial mating via conjugation
plasmid transfer coupled with DNA replication
