Lecture 12 Flashcards
How do viruses defy the central dogma? (2)
1) reverse transcription (Making DNA from DNA)
2) RNA replication
What can PCR tell you about DNA?
tells you how much energy you need to melt the bonds, knowing CG content is important
How does DNA replication work in bacteria?
Starts at origin of replication (oriC) and DnaA binds to and melt the two DNA strands. Replication occurs bi-directionally at the replication forks. DNA replication ends at the terminator (terC)
How does DnaA initiate replication?
DnaA-ATP binds to “DnaA boxes” (DnaA-binding sites) scattered along the oriC
DnaA-ATP molecules hydrolyze ATP to energize their polymerization forming a helical filament
The helical filament exerts torsion on the DNA, melting the origin’s AT-rich region to form a bubble —-easier to break, less H-bonds
What does Helicase DnaB do?
Helicase DnaB is loaded onto the single strands and starts moving to unwind the DNA in the 5’-3’ direction
What does DnaB helicase recruit?
three primase subunits (DnaG)
What does DNA polymerase III do?
enzyme are assembled onto the leading and lagging strands
The Pol III enzyme forms is shaped like a hand, gripping DNA and catalyzing the addition of dNTPs
What direction are the 2 strands replicated in?
5’-3’ direction
What type of supercoils develop as DNA unwinds?
positive supercoils
What relieves supercoiling?
DNA gyrase
What does Tus do?
Tus (terminator utilization substance) binds ter sites and inhibits helicase, halting the replisome
replisomes will run into each other
What type of proofreading activity does Pol III have?
built-in proofreading activity: 3’-5’ exonuclease activity
Prevents Pol III from advancing unless there is a paired nucleotide behind it
What is the MMR pathway?
plan B for catching mismatch pairs, methyl-directed mismatch repair
What does MutS do?
scans the DNA for a mismatch
Recruits MutL and MutH to the mismatch
What does does Mut complex do?
finds a hemi-methylated GATC where MutH cuts the new, non-methylated strand
What does hemi-methylated mean?
one strand methylated (old strand) the other (new strand) is not
What happens if any of the Mut proteins is defective?
there will be high mutation rates
What do restriction enzymes do?
how bacteria cleave unmethylated DNA
How does transcription differ in prokaryotes?
Prokaryotes have one DNA-dependent RNA polymerase: RNAP
What does RNAP make?
all forms of RNA: rRNA, tRNA, small RNAs, and mRNA
What does polycistronic mean?
they encode multiple proteins (usually involved in the same function) in operons
Does Core RNAP have a high or low affinity for DNA?
low affinity
Transcription initiation requires association of RNAP with a subunit called the?
sigma (σ) factor, makes RNAP help like DNA better
What is the role of sigma (σ) factor?
can now bind to the promoter (closed complex)
Once RNAP clears the promoter, it releases the σ-factor and can begin elongation
When does transcription end in prokaryotes?
Transcription ends when RNAP reaches a DNA hairpin called the “termination loop”
The hairpin stalls RNAP and the transcript breaks loose with help of Rho factor
What is the stability and lifespan of mRNA?
mRNAs are unstable and are therefore short-lived
Average half-life of mRNA in E. coli is 1 minute at 37°C
What are Aminoacyl-tRNA synthetase?
charge of matching a particular amino acid to the anticodon of a cognate tRNA molecule
How does Aminoacyl-tRNA synthetase work? (3)
Step 1: the enzyme binds amino acid (1 per enzyme) and reacts it with ATP to form enzyme-bound amino acid-AMP
Step 2: a cognate tRNA binds and is charged with the amino acid releasing AMP
Step 3: after proofreading to ensure the correct aa is attached, the amino acyl-tRNA is released and ready for translation!
What are the 3 stop codons?
Ochre: UAA
Amber: UAG
Opal: UGA
What is the structure of the prokaryotic ribosome?
The prokaryotic ribosome is formed when when the small (30S) and large (50S) subunits come together (70S)
What does the small subunit contain?
recognizes and binds to mRNA and contains the 16S rRNA
What does the large subunit contain?
joins amino acids to make the peptide and contains the 23S and 5S rRNAs
What is the Shine-Dalgarno (SD) sequence?
The 3’ end of the 16S rRNA in the 30S subunit binds to the Shine-Dalgarno (SD) sequence in mRNA
Only mRNAs have this sequence!
What are the 3 tRNA binding sites?
A P E
What does EF-Tu do?
loads aa-tRNA into the the A site using energy from GTP hydrolysis
What happens at the P site?
occupied by the tRNA carrying the growing peptide
What does the 23S rRNA do?
catalyzes peptide bond formation between the new aa and the peptide chain
What happens in termination in prokaryotes?
There are no tRNAs for the stop, or nonsense, codons
When a stop codon is reached, the A site remains empty
Gives time for the ribosome to release factors that enter the A-site and hydrolyze the peptidyl tRNA thereby releasing the completed peptide
