Lecture 5 Stress Responses Flashcards
Importance of microbial adaptation
Stresses:
- Reactive oxygen and nitrogen species
- Acidic pH
- Antimicrobial peptides
- Bile salts
- Extensive damage to bacterial DNA, protein and/or cell envelopes
Nutrient limitation (incl. metals) Hypoxia Extent of nutrient/oxygen limitation varies tremendously within host; impairs bacterial growth & survival
Heat
Shift from ambient temp to host body temp is important signal for pathogens
Adaptation to environment key to causing infection
Strategies for microbial adaptation
Complex regulatory networks (often interconnected) control microbial stress adaptations
Differential recruitment of sigma factors
sigma factor 70 is the ‘housekeeping’ sigma factor
stresses cause the alteration of utilisation of sigma factors (e.g 54) brings about global change in gene expression that allows adaptation
ppGpp
Anti-sigma factors
Adaptor/anti-adaptor proteins
Sigma factors & bacterial transcription
Transcription produces an RNA chain identical in sequence to the coding strand of DNA
Sigma factors bind to RNA polymerase and allow transcription
The key players in transcription
Core RNA polymerase
Sigma factor
Promoter
Promoter is the region of DNA within the genome that contains the consensus sequences that the RNA polymerase complex recognises, binds to, and initiates transcription.
The actual synthesis of the RNA transcript is accomplished by the enzyme, RNA polymerase. The sigma factor forms a complex with the RNA polymerase to facilitate the initiation of transcription
Bacterial RNA polymerase - α2ββ’ω
This single RNA polymerase enzyme is responsible for making all types of RNA
You will usually see the enzyme structure referred to as alpha-2-beta-beta-prime. In some texts, a third subunit is referred to – the omega subunit (RpoZ).
So, Rpol contains two identical alpha subunits – alpha 1 and 2, encoded by the rpoA gene. The alpha subunits play an essential role in the assembly of the core enzyme, in recognizing the DNA promoter regions and in interactions with transcriptional regulators.
It is the beta and beta-prime subunits, encoded by the rpoB and rpoC genes respectively, which are actually the catalytic centres of the Rpol – they are responsible for the synthesis of the RNA transcript.
The omega subunit has long been known to co-purify with Rpol, but for along while no function could really be ascribed to the omega unit. It is now known that omega aids the proper folding and recruitment of the beta-prime subunit to the core RNA polymerase.
So this structure here is what we refer to as the ‘core enzyme’
Promoter recognition by RNA polymerase
There are approx ~13,000 Rpol molecules per Escherichia coli cell
Not all will be engaged in transcription, but almost all are bound (specifically or non-specifically) (electrostatic interaction) to DNA
The “core enzyme” (α2ββ’ω) has general affinity for DNA, primarily through electrostatic interactions
But the core enzyme cannot initiate transcription
The recruitment of a sigma factor (σ factor) is ESSENTIAL for promoter recognition and transcription initiation
Forms the “holoenzyme”
Reduces the affinity for non-specific binding to DNA
Confers specificity for promoter sequences
Sigma factors recognize promoter sequences
Sigma factors are required for the initiation of transcription at promoter regions. Without it, the core enzyme cannot initiate transcription.
RNA polymerase, with sigma factor recruited, makes contact with the DNA; showing the distinct association between different regions of the sigma factor and different features of the promoter region.
-10 and -35 regions is where the sigma factor interacts with the DNA (nucleotides prior to the beginning of the gene)
Different sigma factors recognise different recognition sequences, e.g. sig70RpoD, -35, major sigma factor for normal growth (regulates 1000 genes)
e.g. sig54, nitrogen assimilation genes
sig32, heat shock response
Sigma factors of Escherichia coli
It is important to stress that there is more than one sigma factor in bacteria. Alternative sigma factors can be recruited to the Rpol, resulting in expression of a different subset of genes in order to adapt to a particular environmental stimulus, or stress response.
This table highlights the sigma factors of E. coli.
Sigma-70 is the main sigma factors responsible for expression of most genes, and you can see the promoter consensus sequence for sigma-70 is as we have discussed in the previous slides. However, looking down the table, you’ll see that different sigma factors recognize different consensus sequences at promoter regions, and this obviously explains how they regulate a specific set of genes to cope with a particular environment.
Differential recruitment of σ factors
Sigma factors recognize different promoter consensus sequences
Recruitment of “alternative sigma factors” to transcriptional machinery results in activation of different sets of promoters
This is central to numerous stress responses in bacteria that promote growth & survival in adverse conditions, for example:
Heat shock response Envelope stress response
The Stringent Response – an overview (1)
“Shift down” refers to the transition from an environment with an amino acid excess to amino acid limitation
e.g. from rich medium to defined medium with single C-source
Synthesis of rRNA & tRNA ceases almost immediately
The Stringent Response – an overview (2)
“Shift down” is followed by a process of adaptation
No new ribosomes are produced, and protein & DNA synthesis is curtailed (restricted to essential processes)
Biosynthesis of new amino acids is activated
Biosynthesis of new amino acids is required to compensate for the lack of available amino acids in the external environment. Proteins must be made to synthesize these amino acids, but these proteins are made by the existing ribosomes.
tRNA and rRNA is much more stable than mRNA
The Stringent Response – an overview (3)
Eventually, this adaptive response facilitates resumption of rRNA synthesis production of new ribosomes
Growth resumes, albeit at a reduced rate
This adaptive process is the “stringent response” – mediated by (p)ppGpp
The stringent response
Response to amino acid starvation
Triggered by accumulation of guanosine tetra- and penta-phosphate (p)ppGpp
(p)ppGpp nucleotides called alarmones
Produced by two distinct mechanisms
The stringent response is mediated by guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which collectively are known as (p)ppGpp.
They are ALARMONES - intracellular signal molecules produced in response to harsh environmental conditions. These guanosine nucleotides accumulate rapidly in bacterial cells which are starved for amino acids or exposed to other stress conditions.
The outcome of the stringent response is essentially the shut down of growth and the triggering of an adaptive response that enables survival during stress. Synthesis of ribosomal and transfer RNAs are inhibited, and it profoundly affects cellular processes including transcription, translation and replication. Additionally, this stringent response has been shown to be extremely important for virulence in various bacterial species, including Salmonella.
RelA-dependent (p)ppGpp synthesis (1)
Alarmones are synthesized by the protein RelA, which is found associated with ribosomes (estimated to be approx. one RelA per 200 ribosomes)
Under normal conditions that support translation (with ‘charged’ tRNA molecules), RelA is not active
RelA-dependent (p)ppGpp synthesis (2)
Under amino acid starvation, uncharged tRNAs (that lack an amino acid) accumulate & bind to ribosome, stalling protein synthesis
This triggers RelA to synthesize (p)ppGpp from (GTP)GDP, utilising ATP
Millimolar levels of (p)ppGpp are produced rapidly
(p) ppGpp synthesis triggers release of RelA, which can then act on another stalled ribosome
(p) ppGpp can be synthesized via one of two routes. The first is the RelA-dependent synthesis. During amino acid starvation, uncharged tRNA molecules bind to the acceptor site on ribosomes, stalling protein synthesis. During this paused protein synthesis, RelA (which is found associated with ribosomes) synthesizes (p)ppGpp from GTP or GDP respectively, in a process that utilizes ATP. The abundance of RelA is relatively low (1/200 ribosomes). However, very large concentrations of (p)ppGpp are produced rapidly because the synthesis of (p)ppGpp results in the dissociation of RelA from the ribosome, allowing the RelA protein to shuttle to another stalled ribosome and repeat the process.
(charged tRNA = tRNA + aa)
