Microbiology Chapter 8

E. coli has 3 DNA polymerases
DNA pol I
Plays an essential, albeit secondary role in replication
DNA pol II
Involved in DNA repair
DNA pol III
The major replication enzyme

DNA pol α - synthesis of nuclear DNA
DNA pol β - DNA repair
DNA pol γ - synthesis of mitochondrial DNA
DNA pol δ - synthesis of nuclear DNA
DNA pol ε - DNA repair

-Initiator proteins bind to a specific region of the chromosme termed oriC
-The protein helicase unzips the DNA
-SSBPs stabilize the single-stranded DNA
-The enzyme DNA gyrase relaxes the supercoils generated in front of the replication fork
-The enzyme primase synthesizes a short RNA primer, required for DNA synthesis
-The enzyme DNA pol III synthesizes DNA in the 5’ to 3’ direction continuously on one strand
-On the other strand, short DNA fragments, or Okazaki fragments, are synthesized
-The enzyme DNA pol I removes the RNA primers and replaces them with DNA
-The enzyme DNA ligase seals the gap

RNA pol I transcribes rRNA
RNA pol II transcribes mRNA
RNA pol III transcribes tRNA and 5S rRNA
RNA pol IV transcribes organellar DNA

holoenzyme

Core enzyme
α₂, β, β'
Sigma factor
σ


-The RNA pol holoenzyme binds to the promoter
-It opens the transcription bubble
-After the incorporation of about 10 bases the sigma factor is released
-Transcription continues until a termination sequence (hair-pin structure) is reached

the RNA pol has polymerized 50-60 bases

tRNA 70-95bp
rRNA 120-4200bp
mRNA 100s-100,000s bp

Start AUG GUG UUG
Stop UGA UAG UAA

Tryptophan UGG
Methionine AUG

Mycoplasma
Paramecium
Mitochondria

P (Peptidyl) site = Contains the tRNA with the growing peptide chain
A (Aminoacyl) site = Binds incoming aminoacyl tRNAs
E (Exit) site = Site through which deacylated tRNAs leave the ribosome

1. Initiation
2. Elongation
3. Termination

-The process begins with the formation of a complex between mRNA, the 30S subunit and an N-formyl methionine-tRNA (bacteria) or methionine-tRNA (eukarya/archaea) bound to the P site
-The 50S subunit is recruited to form the complete initiation complex
Specific initiation factors and GTP are required

-An aminoacyl-tRNA enters the A site
-Peptide bond is formed between the two amino acids
-This step is catalyzed by peptidyl transferase
-Translocation of the ribosome moves the growing peptide from the A site into the P site
-The uncharged tRNA leaves the ribosome through the E site
-Specific elongation factors and GTP are required

-The presence of a termination codon in the A site signals the end of the polypeptide
-Release factors bind to the ribosome. GTP is required
-The polypeptide, tRNA, mRNA, and ribosomal subunits dissociate. Everything falls apart.

Transcription
Post-transcriptoin
Translation
Post-translation

transcription

Energy conservation. Saves itself from going any further and wasting energy.

Inducible genes = Genes that are turned on in response to the presence of a small molecule
Repressible genes = Genes that are turned off in response to the presence of a small molecule
Constitutive genes = Genes that are continuously expressed by the cell

repressible

inducible

Structural genes:
lacZ - Encodes β-galactosidase, which cleaves lactose into glucose and galactose
lacY - Encodes β-galactoside permease, which transports lactose into the cell
lacA - Encodes β-galactoside transacetylase, whose function is not well elucidated

Regulatory gene:
lacI - Encodes the lac repressor protein

DNA elements:
lacP - Site where the RNA pol binds
lacO - Site where the lac repressor binds

-Transcription and translation of lacI produces the lac repressor
-The repressor binds to the operator
-Thus, it interferes with binding of the RNA pol to the promoter
==> Transcription of the lac operon genes is OFF

-Allolactose, an isomer of lactose, is the Inducer
-It binds to the repressor ==> repressor changes conformation ==> it loses affinity to the operator and dissociates from it
-Free repressor proteins are also altered so that they cannot bind to lacO
-Therefore, RNA pol can now bind and begin transcription of the Operon
-Transcription and subsequent translation of the β-galactosidase, permease and acetylase genes
==> lactose can now be metabolized

Structural genes:
-Five genes (trpE, trpD, trpC, trpB, and trpA) -> Encode 5 polypeptides that make up 3 proteins that convert chorismic acid to tryptophan
-trpL - Leader region; Encodes the leader polypetide

Regulatory gene:
trpR - Encodes the trp repressor protein

DNA elements :
trpP - Site where RNA pol binds
trpO - Site where the trp repressor protein binds

An Operon is a sequence of adjacent genes that function coordinately under the joint control of an operator and a repressor

Repression = Controls the initiation of transcription
Allows about a 70-fold reduction in the transcription of the operon

Attenuation = Controls the frequency of premature transcript termination
Allows about a 10-fold reduction in the transcription of the operon

The trp repressor exists in an inactive form called the aporepressor
==> cannot bind to trp operator
==> transcription of trp operon
==> trp level increases

Tryptophan (the corepressor) binds to the aporepressor and activates it
==> corepressor-aporepressor complex can now bind to the trp operator
==> RNA pol cannot bind to the promoter to start transcription
==> Operon is repressed

Major groove: 22A
Minor groove: 12A
Complete turn: 34A
Bases spaced at: 3.4A
Diameter: 20A

the precise position of a gene on a chromosome

an alternative form of a gene