Microbiology Exam 2 Flashcards
Bacterial chromosome
Has a circular chromosome
Supercoiled
Archean chromosome
Negative supercoiling
How do bacteria compact their DNA into the cell?
The DNA is supercoiled (twisted) and stabilized by proteins
E. Coli chromosomes
~100 supercoiled domains which are stabilized by proteins
Has DNA gyrase
What is DNA gyrase?
Type of topoisomerase that makes a ds break in the DNA to create supercoils.
Ds DNA passes through the break where it is then religated back together
How does bacterial DNA respond to environmental signals?
Regulation of gene expression
Regulation of protein activity which is:
- faster, why?
- covalent modification, P and de P
- Feedback inhibition
How do proteins bind to specific DNA sequences?
Nitrogenous bases are exposed in major and minor grooves
Each base pair is a unique set of contacts for binding proteins
Contact sites proteins bind to on DNA
G-C
C-G
A-T
T-A
Inverted repeat sequences
Many binding sites have these
Protein repeats (each binds to DNA) are bound by protein dimers
DNA binding domain fits in major groove and along sugar-phosphate backbone
Helix turn helix motif
Present in many DNA binding proteins
Alpha helix can fit in major groove
Amino acid side chains can bind nitrogenous bases (N-bases)
E. Coli genome
88% of it is protein encoding genes (gene dense genome)
70% of the genes are single genes (one gene for mRNA molecule)
Other genes are cotranscribed into a single mRNA molecule (one promoter for multiple genes. Therefore these genes are part of an operon. 6% of operons - 4 or more genes)
Operon
a functioning unit of DNA containing a cluster of genes under the control of a single promoter.
How does sigma subunit work with RNA polymerase?
Initiates txn.
Sigma subunit is required for RNA polymerase to bind to promoter to initiate txn.
sigma subunit 70 in bacterial cells
Default for house keeping genes of E. Coli
For normal growth
E. Coli regulation
Regulation by repressor or activation
can grow in a glucose, minimal salt media
Glucose, ammonia, salts is what E. Coli needs to grow
How is arginine regulated in E. Coli?
When arginine is absent the gene to make it needs to be expressed therefore RNA polymerase binds at promoter which means the default is gene is expressed
Repressor is not bound to operator sequence.
If arginine is present it is a waste of energy to make more and is therefore not going to express the gene
A repressor binds the DNA and blocks gene expression
Does not bind to DNA in the absence of arginine
Does because excess arginine binds to the arginine repressor which then makes the repressor bind to the operator sequence.
When does the Arg repressor protein bind to DNA?
When Arg levels are high and the protein binds Arg
Arg is A.A. required for growth
P-promoter -> size- RNA polymerase binding
O-operator- regulatory sequence (Arg repressor protein binds at operon)
Repressor proteins
Bind to regulatory seq. near the promoter to regulate gene expression
What happens when there are high levels of Arg?
Any repressor protein binds to Arg (corepressor)
Conformational change in the repressor protein
Now binds to DNA at operator sequence
Represents txn.
Lactose Operon
Lactose metabolism
-disaccharide, sugar.
-E. Coli uses it for energy
-Will be catabolized to provide energy
-Broken down to be glucose
Has genes that encode proteins for lactose catabolism
Lac repressor protein
Binds to DNA when lactose is absent
txn. is repressed (very low level of txn. does occur)
Beta-galactosidase
Breaks bond to make galactose and glucose
For Lac operon regulation in E. Coli, when lactose is present
A repressor protein no longer binds to the regulatory region of the gene
Because if you make more while you have some, you are wasting energy
What happens to Lac operon when lactose is present?
Lactose is cleaved by beta-galactosidase to get galactose and glucose by enzyme
Some lactose is converted to allolactose and acts as inducer and allows gene expression
What binds to Lac repressor?
Allolactose
Get change conformation of the repressor protein so that it no longer binds to DNA
Therefore txn. occurs
How will E. Coli metabolize glucose and lactose when both are present?
It will metabolize the glucose first
Then lactose second
Do this because lactose is used to make glucose
Therefore if is already has glucose, then it will use that first to make energy and the lactose after glucose has been depleted
Called graph-diauxic growth
Graph-diauxic growth
There is a lay period after one energy source is depleted
Regulation of the lac operon?
2 proteins important in regulation:
-Lac repressor
-CRP-catabolite responsive protein (cAMP receptor protein):
-DNA binding protein
-Promotes gene expression
-Binds to cAMP=change conformation of protein = binds DNA = promotes gene expression
Adenylate cyclase
Inhibited by glucose
Turns ATP into cAMP and PPi
CRP-cAMP Complex
Binds DNA when there is no glucose
Binds and bends DNA
Changes DNA shape
Therefore RNA pol. can bind to promoter
Get txn.
C site
cAMP binds
P site
RNA pol. binds
O site
lac repressor
Study how Lac operons are regulated
Day 13!
Good Job
For lac operon regulation in E. coli when lactose is present
A repressor protein no longer binds to the regulatory region of the gene
How does blue-white screening?
IPTG:
Chemical analogy of allolactose
Synthetic/not degraded
Binds lac repressor protein.
Therefore beta-galactosidase syn. is not repressed
Becomes white
X-Gal:
Substrate for Beta-galactosidase
Becomes blue
Maltose is a disaccharide that can be that can be catabolized by E. Coli
How is the expression of maltose operon regulated?
An activator protein only binds to DNA in the presence of maltose
There is no repression when maltose is not absent
Maltose operon
Regulated by an activator protein
-Only binds DNA when maltose is present
-Maltose is an inducer
Net result: gene not transcribed. Only transcribed when maltose is present
When glucose levels are low?
cAMP levels are high, CRP does not bind
RNA polymerase of bacterial vs. archaeal?
bacterial RNA-P
sigma factor directs RNA-P to promoter
archaeal RNA-P
similar to eukaryote RNA-P
Has between 11-13 subunits
TATA box
Regulating sequence for archaeal genes
Is bound by TATA binding protein (TBP)
BRE-B recognition element in which it is bound by transcription factor B (TFB)
When these two proteins bind the DNA, they promote the binding of RNA-P
An example of a gene that is regulated in Archaea
Genes involved in nitrogen fixation
(In the Methanococcus genus)
NrpR-repressor protein
Represses expression of genes for nitrogen fixation and glutamine synthesis
What happens when N levels are low regarding N-fixation genes?
alpha ketoglutarate accumulates and binds to NprR protein
This causes a change in conformation in the protein which then no longer binds to DNA
This causes N-fixation genes to be expressed and ammonia is synthesized to turn alpha ketoglutarate to form glutamate
This is because when NprR is releases TBP and TFB can bind to allow DNA-P to bind to DNA
Bacterial genetics format for gene and protein?
Gene: lacZ will be italicized
Protein: LacZ
How is translation initiated in bacteria?
Ribosome binds to the 5’ ribosome binding sites in the start codons
Complementary binding between nucleotides between mRNA & the rRNA molecules
Start codon
AUG
Specifies A.A.: Met
Bacteria: modifies Met as incorporated into the start codon
-formyl-Met: f-Met
Only for start codon
Met is formulated after added onto the tRNA
Attenuation
Transcription regulation-premature termination of transcription
-transcription is initiated but is not completed
Operons that have this (attenuation) have a leader sequence (is before start codon of the first gene) in the mRNA molecule
Leader Sequence
Is translated
Contains codons for the A.A.
Operon codes genes for the biosynthesis
Trp Operon
Leader peptide has 2 trp codons
Are expressed when trp levels are low b/c genes encode synthesis of trp
What happens to Trp operon when trp levels are low?
Not enough trp. low levels of tRNA
leader peptide is not translated and ribosome pauses at Trp codons
Ribosome pauses at the Trp codons
Base pairing at regions 2&3 on the mRNA–> transcription proceeds. Genes expressed so cells can make trp
What happens to Trp operon when trp levels are high?
Trp mRNA has 2 different conformations
Leader peptide is translated because there’s acetylacyl trp for this to occur. Get base pairing between regions 3 & 4 in the mRNA.
Causes termination of transcription. RNA pol. does not transcribe rest of the gene. Can happen because txn. and trans. are coupled and happen simultaneously. Get a loop that forms for it to stop
Transcription stops as the ribosome is able to completely translated to the leader peptide
Get hairpins between groups 3&4 mRNA
This will cause attenuation
Bacterial proteins
Targeted for CM, cell wall or secretion
Their genes code signal sequence = Direct proteins to where to go in cell
Typical signal sequence
15-20 A.A.’s
Located at amino terminus of proton as well
Will have a few basic A.A.
Central region: nonpolar A.A.’s
Followed by polar A.A.’s
