Alternative sigma factors

1
Alternative sigma factors
In certain circumstances, bacteria shut down
normal gene expression and only express a
restricted set of genes. The heat-shock
response occurs when cells are heated to 45C.
Normal gene expression is switched off. Genes
required for refolding denatured proteins are
switched on. Cells deprived of ammonia express
genes required for use of alternative nitrogen
sources.
2
Alternative sigma factors bind to core RNA pol
and direct it to different promoters. E. coli
RNA pol holoenzyme is a2bbs Sigma 70 is used
for normal promoters Sigma 32 is used for
heat-shock promoters Sigma 54 is used for N
limitation promoters
Gene Sigma factor -35 Spacing
10 rpoD s70 TTGACA 16-18 bp
TATAAT rpoH s32 CCCTTGAA 13-15 bp
CCCGATNT rpoN s54 CTGGNA 6 bp
TTGCA
3
The stringent response
When bacteria are starved of nutrients, they
immediately shut down gene expression and
various other metabolic activities. Total RNA
synthesis is reduced to 10 of normal
levels. There is a massive 10 to 20-fold
reduction in rRNA and tRNA transcription. Ribosom
al protein translation decreases. The unusual
nucleotides ppGpp and pppGpp accumulate during
the stringent response.
4
The ppGpp inhibits the elongation phase of
transcription.
The stringent factor RelA is a pppGpp synthetase
that is associated with 5 of ribosomes. RelA
produces one pppGpp every time the A site is
occupied by an uncharged tRNA.
5
Ribosomal protein L11 undergoes a conformational
change when an uncharged tRNA binds. This
activates RelA stringent factor. The SpoT
protein degrades ppGpp so that normal
gene expression can resume rapidly when
conditions improve.
6
Review of gene regulation in bacteria. With
genes that are expressed constitutively,
promoter strength determines the level of
expression. DNA-binding proteins can switch
genes on and off. Repressors switch genes
off. They prevent RNA pol from gaining access to
promoters. Activators switch genes on. They
enable RNA pol to bind to promoters Activity of
repressors and activators can be influenced by
small molecules, temperature, phosphorylation
etc.
7
RNA secondary structure can control gene
expression.
With attenuation, AA-tRNA availability influences
early termination.
With riboswitches, small molecules influence
early termination or translation intiation.
Alternative sigma factors bring about global
changes in gene expression.
The stringent response shuts down gene expression
when times are tough.
Promoter inversion can affect gene expression in
pathogenic bacteria.
8
Brief outine view of translation
GLY
tRNA
anticodon
AAA CCG UUU GGC
5
3
codon
9
There are 20 different aminoacyl tRNA
synthetases. Each one loads an amino acid onto a
tRNA with the correct anticodon.
10
Suppression
Translation of an mRNA for a very short
hypothetical protein.
5 ? AUG - NNN NNN NNN UUG NNN NNN -
UAA ? 3
N ? MET AA AA AA - LEU - AA AA ? C
NNN any codon that specifies an amino acid.
11
Suppression
Nonsense mutation introduces stop codon.
5 ? AUG - NNN NNN NNN UAG NNN NNN -
UAA ? 3
N ? MET AA AA AA ? C
Truncated protein is non-functional.
12
Full-length protein synthesis can be restored by
mutations.
A point mutation can revert back to wild-type UAG
? UUG
Suppressor mutations occur within a different
gene.
13
Suppression
Suppressor tRNA has point mutation in
the anticodon.
TYR
AUC
5 ? AUG - NNN NNN NNN UAG NNN NNN -
UAA ? 3
N ? MET AA AA AA - TYR - AA AA ? C
Full length protein is functional despite LEU ?
TYR change.
14
There are several different tRNA genes for tRNAs
that recognise TYR codons. These can be used to
translate normal TYR codons.
Stop codons UAG Amber UAA Ochre UGA
Opal
15
Extensive suppression of normal stop codons
would give proteins with C-terminal extensions
that are possibly non-functional.
UAG is rarely used as a stop codon in E.
coli. Strains that effectively suppress UAG
nonsense mutations can be isolated.
UAA and UGA suppressors are weak Mutant tRNAs
dont restore much mutant protein
synthesis. Strong suppression of UAA and UGA
would adversely affect general protein synthesis.
16
Conditionally lethal mutations Some mutant
bacterial viruses grow on UAG-suppressing
strains but not on suppressor-free
strains. These have conditionally lethal (CL)
mutations that introduce UAG codons into
essential genes. CL Host range mutations are
lethal in a suppressor-free strain non-lethal
in a suppressing strain.
17
A missense mutation alters an important codon in
a gene. A non-functional polypeptide is
synthesised. Some missense mutations make a
protein abnormally heat-labile. Strains with
these mutations are temperature-sensitive. TS
mutations are also conditionally lethal. TS
mutants grow at a low (permissive)
temperature. die at a high (restrictive or
non-permissive) temperature.
18
Selenocysteine the 21st amino acid
Selenocysteine appears in a number of
oxidoreductase enzymes e. g. formate
dehydrogenase, glycine reductase.
19
The selC gene codes for a tRNA that decodes UGA
UGA
The SelA and SelD proteins convert seryl-tRNA to
selenocysteinyl-tRNA
20
A specialised elongation factor SelB binds to a
selenocysteine insertion sequence SCIS in mRNA.
SelB
UGA
This allows the selenocysteinyl-tRNA to recognise
the appropriate UGA codon.
21
Pyrrolysine the 22nd amino acid
found in enzymes involved in methanogenesis in a
few bacteria and archaebacteria.
Pyrrolysine
UAG
22
A pyrrolysine insertion sequence PYLIS enables
pyrrolysyl-tRNAs to recognise appropriate UAG
codons.
PYLIS
UAG