Genomes, Genes And Operons Flashcards
What is the genome?
The whole of the DNA of an organism.
(. The set of genes
. Additional, non-coding sequences)
What marks the beginning and end of the transcribed part of the gene?
Specific DNA sequences
What does a promoter do?
. Provides binding site for RNA polymerase
. Specifies start point and direction of transcription
What does a transcription terminator sequence do?
Specifies where transcription ends
In eukaryotes how are genes usually transcribed?
Individually
In bacteria how are genes transcribed?
Many genes are arranged into operons- clusters of genes. All the genes in an operon are transcribed from one promoter to give a polycistronic mRNA
What are the genes like in an operon?
They have related functions, and so their products are all needed under the same conditions
What is the start codon that specifies the beginning of translation?
AUG
What does the start codon code for?
Methionine
What are the 3 stop codons that specify the end of translation?
UAG, UGA, UAA
What are the ‘problems’ with start and stop codons?
. Methionine is found in the middle of proteins as well as at the beginning
. In an operon, translation must start at more than one place in an mRNA molecule.
Therefore, cells must be able to distinguish start codons from other methionine codons
How does the cell know whether AUG is a start codon or coding for methionine?
AUG will have AGGAGG next to it ( or something similar)
What is the one gene-one enzyme hypothesis?
Each gene controls the production, function, and specificity of a particular enzyme
What is the typical number of genes in bacteria and in animals?
Bacteria- about 4,000
Animals- about 25,000
In a. Operon where must translation start?
Must start in more than one place in an mRNA (the 5’ end) and moves to first AUG I’m the mRNA. Once it has started transcribing it will carry on until it gets to a stop codon
In bacteria what is the Shine-Dalgamo sequence that the 3’ end of the rRNA binds to?
It is the ribosome binding site in mRNA near the start codon. Sequence is similar to AGGAGG- lines up ribosome so that it knows that at this tart codon it needs to start translating (doesn’t have to be this exact sequence, is usually 2 or 3 bases away from the start codon)
What two conserved regions identify most promoters in bacteria?
-35 and -10 (numbering is from the position at which transcription starts). These are consensus sequence (consensus meaning they may not match this exactly but will be similar)- that bass shown are the commonest but not invariable
What are the five polypeptides in the RNA polymerase core enzyme?
Alpha (2 copies), beta, beta prime, and the weird w one
Specific binging to promotors by the core enzyme requires what?
Sigma factor
What a core enzyme and a sigma factor are combined what does it form?
Holoenzyme
What does the holoenzyme do?
Recognised the -35 and -10 and binds specifically to promotors.
What happens to the sigma factor when transcription starts?
It is left behind (so it is the core enzyme that does the transcribing)
Core enzymes do not know about promotors so what is needed for it to bind specifically to promotors?
An additional polypeptide
Describe bacterial transcription termination
- The end of mRNA folds into a stem-loop structure followed by a succession of uracils. Forms a stem because of complementary sequences close to each other in the DNA sequence and is followed by a sequence of uracils
- This structure destabilises RNA-DNA interaction
- RNA polymerase dissociates from DNA and transcription factors
What do genomes contain?
Genes plus additional DNA
Describe regulation of gene expression in bacteria
A gene must be expressed when it’s product is needed (at the correct level so it doesn’t waste energy). Sequences within an operon specify when it’s genes are expressed. Transcription, RNA stability (e.g. how long it lasts for once it is produced) and translation may all be regulated, sometimes RNA can be produced but it’s does not actually translate them
Why is the function of the lac operon?
To allow E.coil to grow on lactose (sugar isomers glucose and galactose stuck together)
To metabolise lactose, the bacterium E.coil requires two enzymes. What are these and what do they do?
- Lactose permeate- transports lactose into the cell
2. Beta-Galactosidase- hydrolyses lactose to glucose and galactose
What genes are in the lac operon and what do they code for?
. lacZ (z) codes for beta-galactosidase
. lacY (y) codes for lactose permease
. lacA (a) codes for beta-galactoside transacetylase
What are the gene expression requirements for the lac operon?
. Lactose absent or glucose present(Lac operon not transcribed; <5 molecules of beta-galactosidase; dose per cell (so barely transcribed at all)
. Lactose present and glucose absent; lac operon transcribed; about 5,000 molecules of beta-galactosidase per cell
How does the cell recognise whether lactose is present or absent?
Regulation by glucose is a separate process to regulation of lactose
The simple way to detect mutations is to screen for beta-galactosidase activity. How is this done?
Blue colonies contain active beta-galactosidase, white so not. Blue colour produced it or not. Blue colonies contain active beta-galactosidase, white do not. Blue colour produced by action of enzyme on an artificial beta-galactosidase substrate (X-gal). On agar plates, beta-galactosidase can convert the colourless artificial substrate X-gal to a blue product. Thus bacterial colonies containing active beta-galactosidase are blue
See the last page of lecture 3 and the all of lecture 4 (didn’t understand any of it!)
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Describe the structural basis of regulation
No lactose: lac repressor binds tightly to operators because of co-operative binding between subunits. Hinge region holds DNA binding domains in correct orientation to bind operator co-operatively
What causes the repressor to dissociate in the presence of lactose?
When lactose is present, allolactose binds to Lac repressor and causes a conformational change. Hinge region is disordered and subunits do not bind co-operatively to the operator. Changes shape of hinge region so lac repressor falls off
What does a cis-acting (in the same side) mutation affect?
Only affects expression of a gene or operon that is in the same DNA molecule. The regulatory sequence must be attached to the target gene or operon
What does a trans-acting (on different sides) mutation affect?
Affects expression of a gene operon in a different DNA molecule. The regulatory sequence does not need to be attached to the target gene or operon
See bottom of first page and top of second page lecture 5
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Summarise regulation of the lac operon by (allo)lactose
. The lac operon contains genes requires for metabolism of lactose by E.coil
. It is only transcribed when lactose is present
. In the absence of lactose, the tetra metic Lac repressor binds to the DNA of the lac operon at two sites: O1 and O2 or O3
. In the presence of lactose, allolactose binds to Lac repressor and caused a conformational change. Hinge region is disordered and subunits do not bind cooperatively to the operator
The regulation of glucose occurs via cyclic AMP (cAMP). Glucose controls cAMP concentration. How does glucose affect cAMP concentration?
. Glucose present cAMP concentration low
. No glucose: cAMP concentration high. Enzyme adenylate cyclase is activated. cAMP regulates many operon S (>100 promoters)
Give an overview of how regulation of glucose occurs
. Regulation occurs via catabolise activator protein (CAP), which is a dimer. In the absence of cAMP, catabolise activator protein is inactive. cAMP binds to cAP (is a small signal molecule) and cAMP-CAP activates transcription
. Activation is needed because the lac promoter (Plac) is a weak promoter, partly because sequences at -10 and -35 are non-standard
. Demonstration of the importance of the -10 sequence: mutant promoter PlacUV5 does not require activation by cAMP-CAP
Describe the mechanism of glucose regulation
- Catabolise activator protein-cAMP (CAP-cAMP) complex binds to DBA next to -35 region of promoter. Bends DNA by 90 degrees
- RNA polymerase alpha subunit binds to CAP. This increases binding of RNA polymerase to the promoter and so activates transcription (Helps RNApolymerase to bind to alpha-subunit which activates transcription
Give a summary of when transcription of the lac operon is highest
. CAP protein (with cAMP bound) binds to DNA and
. Lac repressor does not bind to DNA
This combination occurs when glucose is absent and allolactose is present. Transcription does not occur if glucose is present or allolactose is absent.
See the examples of operon functions
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How do bacteria sense there is other bacteria (quorum sending)? (Seen in bioluminescence in bobtail squid)
Cell density is measured by quorum sensing, under control of the lux operon, this is what the bacteria use to produce light. Signals of cell density are actually homoserine lactobes (AHL)
- Luxl protein catalyses synthesis of an acyl homoserine lactose (AHL), which diffuses our of cells
- When enough bacteria produce AHL, concentration outside cells becomes high
- Bacteria also take up AHL. When concentration is high outside cells, concentration rises inside cells and AHL binds to LuxR protein (detects the presence of AHL inside the cell)
- LuxR with AHL bound transcription of bioluminescence genes in lux operon
What is the bacteria that bioluminescence to disguise bobtail squids shadows?
Symbiotic Vibro fisheri
See second to last page of lecture 5
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What does AHL bind to?
The LuxR gene
When AHL binds to LuxR what does LuxR bind to and what does this do?
The lux box in DNA next to the lux operon promoter and increases binding of RNA polymerase to the promoters.
What is the luxl gene?
A lux operon
Activation of luxl transcription increases what?
AHL synthesis
Summarise the regulation of transcription in bacteria
- Gene expression responds to environmental signals
- Changes in transcription are an important part of changes in gene expression
- Bacterial genes are typically organised into operons
- Regulation of transcription commonly involves regulatory proteins that bind to specific DNA sequences. These affect the ability of RNA polymerase to bind to promo and initiate transcription
- Regulatory proteins can be depressors or activators
- The ability of a regulatory protein to bind to DNA can change when it: a. Binds a signal molecule b. Is chemically modifies
- Regulatory systems may affect single operons or coordinate the expression of a set of operons
