Bacterial Gene Expression 2 Flashcards
1
Q
trp operon - Genes and Order
A
-the trp operon consists of 5 genes encoding enzymes of the shikimate pathway that converts chorismate to tryptophan (an amino acid)
-the trp operon is only expressed when tryptophan is required in the cell
promoter-operator-trpL—-trpE-trpD-trpC-trpB-trpA
2
Q
Regulation of trp operon at Transcription Initiation Level
A
- repressible operons
- a small molecule, tryptophan, facilitates the binding to DNA of a transcription factor
- the repressor of the trp operon, TrpR, binds to the operon only when it is bound by the co-repressor, tryptophan
- this is the opposite of the control of the lac operon
- tryptophan acts as a co-repressor with TrpR, not an inducer
3
Q
How do we know that there is a second level of regulation of the trp operon?
A
- TrpR reduces transcription of the trp operon by 70x
- but the presence of tryptophan leads to a decrease in the level of the trp operon enzymes by 560-700x
- this means that there must be another level of regulation
4
Q
Second Level of Regulation of the trp Operon
Outline
A
- the second regulatory mechanism is also at the level of transcription but occurs after initiation
- it is called attenuation and causes transcription termination before RNA polymerase can reach the first gene of the operon, trpE
- cis-acting regulatory elements are in the 5’ leader of the mRNA called TrpL, between the 5’ end and the start codon of TrpE
- within trpL is a sequence called the attenuator
- the attenuator contains a short open reading frame that contains 4 segments that can form three alternative secondary structures depending on the levels of tryptophan in the cell
- segment one contains a run of many consecutive tryptophan codons
5
Q
Second Level of Regulation of the trp Operon
Low Tryptophan
A
- if tryptophan levels in the cell decrease
- then levels of tRNAs with tryptophan also decrease
- a ribosome following behind the RNA polymerase will stall when it reaches segment 1 as there aren’t enough tRNAs
- the ribosome is covering region 1
- RNA polymerase continues ahead and transcribes segements 2 and 3
- segment 1 is covered so segment 2 cant bind to it, instead segments 2 and 3 bind
- region 4 is left unpaired and RNA polymerase can continue to transcript the trp operon genes
6
Q
Second Level of Regulation of the trp Operon
High Tryptophan
A
- ribosome doesn’t stall at region 1 as there are enough tryptophan tRNAs
- the ribosome proceeds to the stop codon between segments 1 and 2 and stops partially covering segment 2
- when RNA polymerase synthesises region 3 it cant pair with region 2, instead it pairs with region 4
- the pairing of segments 3 and 4 creates a rho-independent transcriptional terminator
- RNA polymerase doesn’t reach the genes of the trp operon
7
Q
pheA
A
-like the trp operon, uses a repressor to switch ON/OFF expression of the operon and uses attenuation to fine tune expression
8
Q
CREs
A
- cis-regulatory elements
- regions of non-coding nucleic acid that regulate expression of co-localised genes
- found in the vicinity of the genes they regulate
- typically regulate gene expression by functioning as binding sites for other factors, including but not limited to proteins
- cis - on this side
9
Q
Trans-acting Factors
A
- encode diffusible factors that can regulate the expression of genes at a distance
- trans - on a different side
10
Q
Eubacterial Translation Initiation
A
- ribosomes start at the start codon (usually AUG, but sometimes GUG or UUG) within 5-8 bp of the RBS (ribosome binding site on the mRNA
- the RBS has a degree of complimentarity to a segment of 16S rRNA in the ribosome
- similarity to consensus can determine the efficieny of translation
- translation initiation is a major point of gene regulation in eubacteria
11
Q
Regulation at Level of Translation Initiation
A
- ribosomal subunit assembly is highly coordinated
- synthesis of ribosomal proteins never exceeds that which can be assembled with RNA to produce complete translational machinery
- underlying mechanism of auto-regulation of translation
- if
12
Q
Regulation of Translation Without Protein Binding - prfA
A
- in human pathogen Listeria which causes food poisoning
- virulence genes are only expressed at 37C (human body temperature)
- transcription factor regulating these virulence genes is prfA
- expression of prfA is controlled at the level of transcription
- mRNA of prfA has a 5’ leader sequence that can form a secondary structure
- this secondary structure incorporates the Shine-Dalgarno sequence (RBS)
- ribosomes can only access the S-D sequence when the secondary structure melts which occurs at 37C
13
Q
Regulation of Translation by Small RNAs - ompF
A
- ompF gene encodes a major outer membrane porin
- a porin is a non-specific transport that allows the passive diffusion of small, polar molecules
- translation of pre-made ompF mRNA is regulated
- antisense RNA from micF gene is synthesised which is complimentary to ompF mRNA
- the antisense RNA from micF binds to the ompF RNA to prevent translation initiation
14
Q
RNA Regulation Beyond Anti-Sense RNAs
A
- 6S RNA mimics a promoter recognised by sigma 70
- it binds to and inhibits RNA polymerase
- a regulation of transcription initiation
15
Q
Why use sRNAs instead of proteins to regulate gene expression?
A
- RNA world hypothesis - a world filled with RNA-based life which predates current DNA-based life
- RNA can store information like DNA and catalyse reactions like proteins do, may have supported pre-cellular life
- RNA worl evolved into a DNA world
