This preview shows page 1. Sign up to view the full content.
Unformatted text preview: Sigma Factors and Bacterial Transcription
Textbook Reading Pages 352-353 “Steps in RNA Synthesis” The points to be remembered from this text:
Know that there are promoters and terminators that determine the start and end points
Know of what core RNA polymerase is comprised.
Know what is the function of core RNA polymerase.
Know of what holo RNA polymerase is comprised.
Know what is the function of the holo form of RNA polymerase. Textbook Reading Pages 354-355 “Promoters and Sigma Factors” The points to be remembered from this text:
Know what is the function of the sigma subunit of RNA polymerase.
What is the function of the -10 and -35 sequences?
What is the difference between sigma-70 (sigma-A) and alternative sigma factors?
What is an anti-sigma factor?
Know how FlgM inhibition of sigma-28 relieved to allow expression of late flagellar
genes. Stress Response
Many alternative sigma factors are involved in reprogramming gene expression in response to an
environmental stress. A stress is defined as an abiotic factor(s) that limits the production of
biomass. Examples of stress factors are unfavorable temperature, pH, water availability, and
Adaptation to Stress
Cells are capable of sensing and responding to stress. Alternative sigma factors are often part of
the signal transduction pathways for responding to stress. A response to stress can be easily
demonstrated by observing that bacteria that are exposed to a sublethal level of a stress factor are
able to survive for longer periods of exposure to a lethal level of that stress factor than cells that
were not exposed to the sublethal level of stress.
Determining proteins induced in response to stress
To determine which proteins are most highly transcribed and translated in cells, cells are
incubated with a radioactive amino acid, 35S-methionine, for a short period of time (e.g. 1
minute). Only proteins that are being synthesized in that short period of time are become
radioactive. After that short period of incubation, lyse (i.e. break) the cells. The proteins of the
cells can then be separated based on size by SDS-PAGE (SDS-polyacrylamide gel
electrophoresis). If the SDS-polyacrylamide gel is exposed to film, you will see the number and
location (i.e. size) of the labeled proteins. Fig. 1 shows proteins that are synthesized during
1 growth of Escherichia coli cells to 42°C, a sublethal temperature stress, as compared to those
synthesized in cells grown at 30°C . Note that only the proteins synthesized at the highest rate
are observed by this method. Additional methods that will not be covered are needed to identify
the proteins that were radiolabelled. Fig. 1. E. coli protein synthesis. Cells were
incubated for 1 minute with 35S-Met and proteins
analyzed by SDS-PAGE.
Chaperones and Proteases
Heat shock proteins (proteins induced by high temperature stress) were found to be chaperones
and proteases. Chaperones aid in the folding of proteins or keep proteins unfolded to be
degraded by proteases. Both chaperones and proteases limit the damaging effects of unfolded
proteins that occur at higher temperatures.
Transcription of Genes for Heat Shock Proteins
To determine whether transcription of genes for heat shock proteins changed in response to an
upshift in temperature, RNA is isolated from cells at various times after shift of cells to 42°C and
prior to the temperature shift. The level of mRNA for a heat shock gene (e.g. groE) was
determined. An example of a technique that is used currently to determine the level of mRNA is
real-time PCR. We will not have time to go into this technique. Know that an important control
in determining the level of a specific mRNA is to have a value to which to normalize the specific
mRNA values. This normalization could be to the total RNA in the sample or the level of
another mRNA that should not change in response to temperature. Fig. 2 shows the level of
groE mRNA of E. coli in cells at various times after temperature up-shift. Note that the groE
mRNA is presented as a % of the total RNA. This figure also shows the level of groE mRNA in
a wild-type strain (WT) and a rpoH mutant strain. 2 Fig. 2
Sigma-32 Directs Core RNA polymerase to transcribed heat shock genes
The rpoH gene was shown to encode a protein with amino acid sequence similarity to the sigma
subunit of RNA polymerase. The rpoH gene product is called sigma-32. The sigma normally
associated with RNA polymerase is called sigma-70. To test the hypothesis that sigma-32 was
alternative sigma factor that would direct transcription of heat shock genes, an in vitro
transcription assay was used as follows:
Step 1. Incubate in a test tube:
a) Purified E. coli RNA polymerase (Holoenzyme that includes sigma-70 or Core Polymerase)
b) ATP, GTP, CTP, and 32P-UTP (the radioactive nucleotide is critical)
c) Plasmid DNA that has a promoter for a heat shock gene. The plasmid DNA is cut with a
restriction enzyme a ~100 bp downstream of the start site for the heat shock gene promoter.
Step 2. Terminate reaction and separate RNA from protein by treating the above solution with
organic solvents (phenol and chloroform). Separate the RNA species on a polyacrylamide gel
and expose the gel to film to visualize the radioactive RNA. Fig. 3. 3 Fig. 3 shows an in vitro transcription assay with sigma-32. Note that neither holo RNA
polymerase nor core RNA polymerase transcribes the heat shock promoter (lanes 1 and 4
respectively). Core RNA polymerase with sigma-32 transcribed the heat shock promoter (lane
Sigma-28 Direct Transcription of Flagellar Genes.
Early and middle flagellar genes are required for assembly of the hook and basal body of the
flagellum. One of these middle flagellar genes is the fliA gene, which encodes the alternative
sigma-28 and which is required to direct transcription of late flagellar genes. If a mutation is
made in one of the early/middle flagellar genes (fla-) that prevents assembly of the hook or basal
body, then late flagellar genes are not expressed even if sigma-28 is present in cells. Expression
of the late flagellar genes was measured using an in vivo transcription assay (fljB-lacZ), which is
FlgM, the anti-sigma factor for sigma-28.
The expression of late flagellar gene expression in a fla- mutant is enhanced by a mutation that
disrupts the function of the flgM gene. To understand the mechanism by which FlgM normally
prevented transcription of the late flagellar genes, the FlgM protein was purified. Purified FlgM
was added to an in vitro transcription assay with Core RNA polymerase and sigma-28. FlgM
specifically inhibited transcription of sigma-28 dependent genes and not sigma-70 dependent
genes. The gene for FlgM is an early flagellar gene and thus present when sigma-28 is
synthesized to inhibit its activity. The question from the data was how on assembly of the hook
and basal body was FlgM’s inhibition of sigma-28 release to allow expression of late flagellar
genes. The model that was developed was that FlgM was exported to the outside of the cell
through the assembled hook and basal body. To test this, the level of FlgM was measured in the
extracellular medium by western blot with anti-FlgM antibodies (see below for Western Blot
protocol). FlgM was present in the extracellular medium of wild-type cells, but not in cells that
were defective for assembly of the hook and basal body. 4 In vivo Transcription Assays
Purpose: To monitor the level of transcription of a particular gene in a cell. Generally, the level
of transcription of a gene under two different growth conditions or between wild-type and mutant
strains is compared.
The easiest method to measure transcription of a gene in a bacterial cell is to fuse the promoter of
your gene X to a reporter gene, such as lacZ, and measure the amount of the product of the
reporter gene. lacZ is used as a reporter gene because its product, ß-galactosidase, is easy to
detect due to its ability to cleave substrates such as O-nitrophenol-ß-D-galactoside (ONPG).
Cleavage of ONPG by ß-galactosidase to ONP and G results in a yellow color. This yellow
color can easily be measured using a spectrophotometer. The yellow color is thus proportional to
the level of transcription of the your gene X.
Step 1. Create a DNA fusion between the promoter of your gene X and the coding region of lacZ.
This is a transcriptional fusion. Transcription of lacZ is controlled by the gene X
promoter. However, translation of the lacZ remains under the control of the lacZ
ribosome binding site. 5 Step 2. Introduce the geneX-lacZ transcriptional fusion into the bacterial strain in which you
want to study gene X regulation.
The important point here is that the fusion needs to be on DNA that is replicated so that
the fusion will be maintained in the bacterial cells as they grow and divide. Thus, the
geneX-lacZ transcriptional fusion can be placed on the chromosome or on a plasmid. An
example of a plasmid with the geneX-lacZ fusion can be see in the figure below.
Step 3. Grow bacterial strains containing geneX-lacZ fusion under the desired growth conditions.
Step 4. At desired times, remove an aliquot of the bacterial cells. Determine the number of cells present in the aliquot (e.g. measure turbidity of solution).
Step 5. Disrupt the membranes of the cells with a small amount of an organic solvent (e.g.
chloroform) and detergent (e.g. sodium dodecylsulfate). By disrupting the membranes,
the ONPG, which will be added in Step 5, can diffuse to the ß-galactosidase that is inside
Step 6. Add ONPG to the disrupted cells from Step 4. When you see a yellow color develop, due
to cleavage of ONPG by ß-galactosidase, quantify the amount of yellow color by
measuring the optical density at 420 nm (OD420) of the solution in a spectrophotometer.
Also record the length of time that the cells were incubated with the ONPG before
reading the OD420.
Step 7. Determine the specific activity of ß-galactosidase =
OD420(i.e. amount of yellow color)/(time of incubation with ONPG x # of cells) 6 Western Blots
Purpose: Generally used to detect the amount of a specific protein present in cells. It can be used
to compare the amount of Protein X present in two different strains of bacteria (e.g. a wild-type
strain and a mutant strain).
Necessary Reagent: Antibodies that bind to Protein X. These are often referred to as antiProtein X antibodies.
Step 1. Grow cells to be tested under the desired conditions.
Step 2. Separate cells from culture supernatants by centrifugation.
Step 3. Resuspend cell pellets in solution containing sodium dodecylsulfate (SDS) and heat to
80°C. This will lyse the cells and release their proteins into the surrounding solution.
Step 4. Separate the proteins of the cell based on their size using SDS-polyacrylamide gel
electrophoresis (SDS-PAGE). Note: the SDS in the gel binds to the proteins, keeps the
proteins denatured, and gives them a negative charge. All proteins will move towards
the positive electrode. The polyacrylamide gel will filter the proteins such that the
largest proteins remain at the top of the gel, the smaller ones at the bottom.
Step 5. Transfer the proteins from the gel to a filter, such as nitrocellulose. Applying an
electrical current across the gel so that the proteins, which will move towards a positive
charge, move towards the nitrocellulose can do this. The proteins will stick to the
nitrocellulose, as proteins like to do this.
Step 6. Block all free surfaces that can bind protein on the nitrocellulose with a non-specific
protein like bovine serum albumin (BSA). This is done by incubating the nitrocellulose
membrane from Step 5 with a solution containing BSA. If this is not done, then in Step
7, the Anti-protein X antibody, which is a protein, will bind to the nitrocellulose. You
only want Anti-protein X antibody to bind to Protein X, not the nitrocellulose.
Step 7. Incubate the nitrocellulose from Step 6 with a solution of Anti-Protein X antibody to
allow the antibody to bind Protein X on the nitrocellulose.
Step 8. In order to detect where on the nitrocellulose the anti-Protein X antibody bound,
incubate the nitrocellulose filter from Step 7 with a solution containing a secondary
antibody that binds to the anti-Protein X antibody (i.e. Primary Antibody). The
secondary antibody is conjugated to Horse Radish Peroxidase (HRP). In the presence of
the appropriate substrates, the HRP can lead to the production of light (see figure
below). This light can be detected by exposing the nitrocellulose filter to a photographic
film. 7 Note: False positive results will be obtained if the anti-Protein X antibody binds to proteins other
than Protein X. To be sure that this is not the case, test that the anti-Protein X antibody
does not detect any protein from mutant cells that lack Protein X. 8 ...
View Full Document
This note was uploaded on 03/06/2012 for the course MIMG 100 taught by Professor Lazazzera during the Summer '10 term at UCLA.
- Summer '10