Lecture 12

Question 1

How do viruses defy the central dogma? (2)

Answer 1

1) reverse transcription (Making DNA from DNA)
2) RNA replication

Question 2

What can PCR tell you about DNA?

Answer 2

tells you how much energy you need to melt the bonds, knowing CG content is important

Question 3

How does DNA replication work in bacteria?

Answer 3

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)

Question 4

How does DnaA initiate replication?

Answer 4

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

Question 5

What does Helicase DnaB do?

Answer 5

Helicase DnaB is loaded onto the single strands and starts moving to unwind the DNA in the 5’-3’ direction

Question 6

What does DnaB helicase recruit?

Answer 6

three primase subunits (DnaG)

Question 7

What does DNA polymerase III do?

Answer 7

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

Question 8

What direction are the 2 strands replicated in?

Answer 8

5’-3’ direction

Question 9

What type of supercoils develop as DNA unwinds?

Answer 9

positive supercoils

Question 10

What relieves supercoiling?

Answer 10

DNA gyrase

Question 11

What does Tus do?

Answer 11

Tus (terminator utilization substance) binds ter sites and inhibits helicase, halting the replisome
replisomes will run into each other

Question 12

What type of proofreading activity does Pol III have?

Answer 12

built-in proofreading activity: 3’-5’ exonuclease activity
Prevents Pol III from advancing unless there is a paired nucleotide behind it

Question 13

What is the MMR pathway?

Answer 13

plan B for catching mismatch pairs, methyl-directed mismatch repair

Question 14

What does MutS do?

Answer 14

scans the DNA for a mismatch

Recruits MutL and MutH to the mismatch

Question 15

What does does Mut complex do?

Answer 15

finds a hemi-methylated GATC where MutH cuts the new, non-methylated strand

Question 16

What does hemi-methylated mean?

Answer 16

one strand methylated (old strand) the other (new strand) is not

Question 17

What happens if any of the Mut proteins is defective?

Answer 17

there will be high mutation rates

Question 18

What do restriction enzymes do?

Answer 18

how bacteria cleave unmethylated DNA

Question 19

How does transcription differ in prokaryotes?

Answer 19

Prokaryotes have one DNA-dependent RNA polymerase: RNAP

Question 20

What does RNAP make?

Answer 20

all forms of RNA: rRNA, tRNA, small RNAs, and mRNA

Question 21

What does polycistronic mean?

Answer 21

they encode multiple proteins (usually involved in the same function) in operons

Question 22

Does Core RNAP have a high or low affinity for DNA?

Answer 22

low affinity

Question 23

Transcription initiation requires association of RNAP with a subunit called the?

Answer 23

sigma (σ) factor, makes RNAP help like DNA better

Question 24

What is the role of sigma (σ) factor?

Answer 24

can now bind to the promoter (closed complex)
Once RNAP clears the promoter, it releases the σ-factor and can begin elongation

Question 25

When does transcription end in prokaryotes?

Answer 25

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

Question 26

What is the stability and lifespan of mRNA?

Answer 26

mRNAs are unstable and are therefore short-lived Average half-life of mRNA in E. coli is 1 minute at 37°C

Question 27

What are Aminoacyl-tRNA synthetase?

Answer 27

charge of matching a particular amino acid to the anticodon of a cognate tRNA molecule

Question 28

How does Aminoacyl-tRNA synthetase work? (3)

Answer 28

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!

Question 29

What are the 3 stop codons?

Answer 29

Ochre: UAA Amber: UAG Opal: UGA

Question 30

What is the structure of the prokaryotic ribosome?

Answer 30

The prokaryotic ribosome is formed when when the small (30S) and large (50S) subunits come together (70S)

Question 31

What does the small subunit contain?

Answer 31

recognizes and binds to mRNA and contains the 16S rRNA

Question 32

What does the large subunit contain?

Answer 32

joins amino acids to make the peptide and contains the 23S and 5S rRNAs

Question 33

What is the Shine-Dalgarno (SD) sequence?

Answer 33

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!

Question 34

What are the 3 tRNA binding sites?

Answer 34

A P E

Question 35

What does EF-Tu do?

Answer 35

loads aa-tRNA into the the A site using energy from GTP hydrolysis

Question 36

What happens at the P site?

Answer 36

occupied by the tRNA carrying the growing peptide

Question 37

What does the 23S rRNA do?

Answer 37

catalyzes peptide bond formation between the new aa and the peptide chain

Question 38

What happens in termination in prokaryotes?

Answer 38

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