Microbial Gene Expression And The Central Dogma (part 3) Flashcards
Expression of genes is an ________________ intense process (especially for __________________).
Energy
Prokaryotes
What is spent during the expression of genes in prokaryotes?
1 nucleotide triphosphate (ATP, GTP, CTP, UTP) spent per nucleotide of RNA during transcription
2 GTP spent per amino acid in a protein during translation (elongation process)
Microbes typically have _______________ of genes, but only a ______________ f them need to be expressed at any given moment.
Thousands
Small subset
What are used to increase or decrease the rate of transcription or translation? Give an example
Environmental signals.
E.g genes that metabolize the sugar, lactose, are only produced when lactose is present in the environment
Certain types of genes are always expressed regardless of the environmental signals. What are they called and and what is their control system called?
Housekeeping genes
Constitutive gene expression
Explain inducible and repressible genes. They are a form of _____________________ that controls most genes.
Inducible genes: typically not expressed but their expression can be activated by environmental signals.
Repressible genes: typically expressed but their expression can be inhibited by environmental signals.
Regulated expression
What are the 2 types of regulated gene expression systems?
- Transcriptional controls are systems that induce or repress the rate of transcription of a gene into mRNA
- Translational controls are systems that induce or repress the rate of translation of an mRNA into a protein.
Most types of transcriptional controls depend on the use of proteins called ______________________. Which work by enhancing or disrupting the ______________________ of transcription.
Transcription factors
Initiation
Initiation of transcription depends on a complex interaction between what 2 things?
Sigma factor and RNA polymerase at the gene’s promoter.
How can transcription factors increase and decrease transcription of a gene?
Increase by enhancing and stabilizing the interaction between the sigma factor and the RNA polymerase. (Transcriptional activators)
Decrease by inhibiting and destabilizing the interaction between the sigma factor and the RNA polymerase. (Transcriptional repressors)
How do transcriptional activators stabilize RNA polymerase?
By binding to activator binding sites upstream of the promoter.
How do transcriptional repressors destabilize or block RNA polymerase?
By binding to operator sequences that are downstream or overlap with the promoter.
What is the trp operon and example of? (2 things)
Transcriptional control and a repressible genes.
What is the trp operon partially regulated by?
A protein called the trp repressor, which is made by a separate gene elsewhere on the chromosome.
What does the trp repressor bind to when active? What is it downstream of?
The trp operator and the promoter, which is where the sigma factor binds.
Under what conditions is the trp operon activated?
When there is little or no tryptophan.
What do low levels or tryptophan mean?
None is available to bind to the repressor. Without tryptophan bound to it, the repressor is inactive and unable to bind to the operator. As long as the repressor is inactive, transcription is initiated and trp genes are made into mRNA and proteins.
What happens when there is lots of tryptophan in the environment?
The trp operon will be repressed and the amino acid tryptophan will bind to the trp repressor and activate it. As long as tryptophan is attached to the repressor, the repressor can bind to operator and block transcription initiation.
Because the binding of tryptophan is required for repression, what is tryptophan itself called?
A co-repressor
What is leakage expression?
When some mRNA still gets made after a repressor blocks transcription.
The lac operon is an example of what 2 things? What does it encode?
Transcriptional controls and an inducible gene.
Encodes 3 enzymes needed to metabolize lactose as a source of carbon.
The lac operon uses a combination of both _____________________ and __________________.
Transcriptional activation and repression.
What is the activator binding site on the map labeled for the lac operon?
CAP Site
What happens when the lactose levels are low?
The lac operon is repressed.
What happens in the absence of lactose from the environment?
Lac repressor is active and binds to the operator sequence which blocks the ability of RNA polymerase to bind to the promoter. When repressed, very little mRNA is made.
What happens to the cells of E. Coli in the repression conditions?
The cells only have 3 molecules of each lac operon protein due to leakage expression.
What happens when lactose levels are high in the environment?
The lac operon is partially induced.
What happens to some lactose in microbial cells?
Some is converted into a similar molecule called Allolactose.
What does Allolactose bind to?
It binds to the lac repressor, making the repressor inactive and unable to bind to the operator. As long as the Allolactose is attached to the repressor, RNA polymerase can initiate transcription.
What is needed to fully induce transcription of the lac operon? What do heterotrophic cells prefer?
A second protein, catabolite activator protein (CAP) , which is only active when glucose levels in the cell are low.
Glucose
Operon like lac are only fully induced if the cell does not have enough ________________.
Glucose
What happens when glucose levels are low?
CAP becomes a transcriptional activator of the lac operon.
What does the binding of CAP to the DNA CAP site do?
Stabilizes RNA polymerase and increases the transcription of lac genes.
What is Arabinose? What is the abbreviation/name of the Arabinose operon?
Arabinose is a sugar that can be metabolized by feeding it into the pentose phosphate pathway (PPP).
(ara)
What is unusual about Arabinose?
It can be both repressed and activated by the same protein called AraC.
What does the AraC bind to? (2)
2 DNA sequences:
1. The araO2 sequence is the operator sites
2. The araI sequences are the inducer sites
What happens when Arabinose is absent?
The operon is repressed and the AraC proteins will bind together across the operator and inducer. This causes the DNA to physically loop around the gene (steric effects).
What does the DNA loop of AraC proteins do?
Interferes with RNA polymerase binding and prevents transcription. It also blocks access to the Arabinose promoter (ParaBAD)
What is special about the repression mechanism of the Arabinose operon? What is it often used in?
Has a very low leakage expression.
Used in genetically engineered plasmids like pGLO
What happens when Arabinose is present?
The operon is partially induced.
What does Arabinose bind to?
The AraC proteins which changes the way they interact with each other.
Once Arabinose is bound to AraC, what happens?
AraC proteins dimerize and bind only to the inducer sites, which stabilizes RNA polymerase and increases transcription of the operon.
What is the ara operon fully induced by?
Activated CAP protein.
When is the maximum amount of expression of the ara operon?
When Arabinose levels are high and glucose levels are low.
What can interrupt transcription elongation? And what is the mechanism of interruption called?
Some types of transcriptional controls.
Transcription attenuation
In addition to its repressor that regulates initiation of transcription, the_________________ is also regulated during elongation.
Trp operon
What is the regulation of the trp operon during elongation due to?
A special sequence in its leader (labeled trpL) called the attenuator.
What can the attenuator do? What does it help?
It can interrupt transcription if tryptophan levels are high.
Mechanism is one way to deal with the problem of leak expression.
The attenuator consists of 4 ________________ that can base pair with each other to form _____________________.
RNA sequences
Stem loop structures
What does the first sequence of the four RanA sequences of the attenuator contain?
2 codons for tryptophan
What must happen for the transcription of trp? What happens if it doesn’t?
Sequences 2 and 3 of the attenuator must form a stem loop, called the anti-terminator loop.
Sequences 3 and 4 will form a stem loop instead called the terminator loop, which terminates transcription.
What is a key fact to remember when thinking about the transcription attenuation?
Is that in microbes, mRNA molecules are translated by the ribosome while they are still being transcribed by RNA polymerase. This means that the ribosome can potentially interact with the RNA polymerase.
What happens if tryptophan levels are low?
The cell needed to express the trp operon to make tryptophan. In this case, the ribosome that is translating the mRNA becomes stalled at the tryptophan codons in sequence 1.
Why does the mRNA become stalled is tryptophan levels are low?
There won’t be enough tryptophan tRNA to quickly translate the leader so the ribosome stops while it waits for tryptophan tRNA.
What does stalling of the ribosome allow?
Sequences 2 and 3 to form the anti-terminator loop and the elongation of the mRNA continues and trp proteins get made.
What happens if tryptophan levels are high?
The cell needs to repress trp and the ribosome that is translating the mRNA does not stall a the the tryptophan codons in sequence 1.
Why does the ribosome that is translating the mRNA not stall?
Because the tryptophan levels are high and there is plenty of tryptophan tRNA, so the ribosome keeps translating the leader. This blocks the formation of the 2-3 loop and we get the 3-4 terminator loop formation instead.
What does the terminator loop do?
Will pull the mRNA out of the RNA polymerase exit port and results in transcription terminator.
