Protein Synthesis and DNA Replication - Genes

Question 1

What is DNA replication and how often foes it occur in a cell cycle?

Answer 1

Producing two identical DNA molecules from one DNA molecule - it must happen only once per cell cycle

Question 2

What is the enzyme that synthesizes DNA and in what direction does it do this?

Answer 2

DNA polymerase III (Pol III) - 5’ to 3’ direction

Question 3

What does highly processive mean when referring to Pol III?

Answer 3

Once it starts the enzyme doesn’t dissociate until it is terminated

Question 4

What is an Okazaki fragment and why are they formed?

Answer 4

Pol III has no issue continuously replicating the leading strand as it is in the 5’ to 3’ direction. However the lagging strand is in the 3’ to 5’ direction so a series of ~1500 base fragments are replicated in the 5’ to 3’ direction before being joint togther

Question 5

What does semi-discontinuous DNA replication refer to?

Answer 5

The leading strand is replicated continuously but the lagging strand is replicated in Okazaki fragments discontinuously

Question 6

What is a primer?

Answer 6

A piece of RNA that is used as a starting point as Pol III can not begin synthesis alone. For the leading strand only one is require but many are required for the lagging strand

Question 7

What is the enzyme that is responsible for making primers?

Answer 7

DNA primase

Question 8

What are the functions of DNA polymerase 1 and DNA ligase?

Answer 8

DNA polymerase 1 - replaces RNA primers with DNA
DNA ligase - joins the newly replicated DNA on the lagging strand together

Question 9

What is the function of DNA helicase?

Answer 9

It unwinds duplex DNA to single stranded DNA

Question 10

What does in mean if DNA is positively supercoiled?

Answer 10

It is overwound

Question 11

What is a linking number and what is its equation?

Answer 11

The number of times one strand winds around the other
Lk = Lk0 = N/h (for relaxed DNA)
Where:
N = number of base pairs
h = 10.5 (helical repeat in solution)

Question 12

What is the equation for specific linkage difference (σ)?

Answer 12

σ = ΔLk/Lk0 where ΔLk = Lk - Lk0

Question 13

Does in vivo DNA have a higher or lower linking number than in vitro?

Answer 13

DNA in cells is negatively supercoiled (underwound) and so it has a lower linking number than synthesized DNA

Question 14

What are DNA topoisomerases?

Answer 14

Enzymes that break closed circular DNA to resolve topological complexity (over- or underwound DNA) and can pass remote DNA through the break

Question 15

How can the linking number of a closed circular DNA be changed without breaking the DNA?

Answer 15

It can’t !!!!

Question 16

What is the initiation of DNA replication called?

Answer 16

oriC

Question 17

What protein binds to DNA for replication to begin?

Answer 17

DnaA

Question 18

In which direction does DNA replication occur?

Answer 18

It is bidirectional

Question 19

What are the steps involved in DNA replication initiation?

Answer 19

1) Binding of DnaA to DnaA boxes at oriC
2) Unwinding of DNA double helix by a DNA helicase
3) DNA primase synthesizes RNA primers
4) Pol III extends RNA primers with DNA

Question 20

What are the “ingredients” used in Sanger sequencing of DNA?

Answer 20

• single stranded DNA template (usually from a plasmid)
• primer
• DNA polymerase (Pol III)
• dATP, dCTP, dTTP, dGTP
• radioactive dATP
• one of ddATP, ddCTP, ddTTP, ddGTP (for termination)

Question 21

Why does ddATP result in termination?

Answer 21

The missing the 3’ OH group results in termination and therefore if the template is T and requires an A there is a chance that A will come from ddATP and cause termination

Question 22

How does polyacrylamide gel electrophoresis work?

Answer 22

Negative DNA moves due to an electric current with smaller strands of DNA moving more quickly towards the positive charge. They are read by moving up and seeing which base is next

Question 23

In which direction do peptides grow?

Answer 23

From the N-terminus to the C-terminus

Question 24

Does DNA replication or translation have a higher error frequency?

Answer 24

Translation has a higher error frequency as the consequence of one protein having an incorrect amino acid is far less than that of DNA having an error which will result in many proteins with errors for many generations

Question 25

What does fidelity mean?

Answer 25

The degree of exactness with which something is copied or reproduced

Question 26

What 2 thing do fidelity in protein synthesis depend on?

Answer 26

1) Accurate ligation of an amino acid to its correct tRNA by aminoacyl-tRNA synthetase
2) Recognition of tRNA to mRNA

Question 27

What are the steps in translation initiation ?

Answer 27

1) binding of ribosome to mRNA
2) tRNA binds to formyl-methionine
3) tRNA binds to mRNA
4) formyl-methionine binds to ribosome

Question 28

What does this mean: “the tRNA anti-codon bonds anti parallel to the codon in mRNA” ?

Answer 28

The anti-codon say; GCA will bind to UGC - not CGU in mRNA

Question 29

Where and what type of bond does the amino acid bond to tRNA?

Answer 29

An ester bond at the 3’ hydroxyl

Question 30

What is the wobble position?

Answer 30

The last base in a codon - GCU, GCC, GCA and GCT all code for analine - tRNA for analine must be able to recognise all these codons

Question 31

What interactions happen at each base during codon - anti-codon recognition?

Answer 31

At the first two positions, Watson-Crick pairings take place between the codon and anti-codon base but and the third position there are fewer ribosome interactions allowing wobble and more than typical Watosn-Crick pairing to take place e.g. U – G or A – C

Question 32

What does the first base in an anti-codon do?

Answer 32

It determines how many possible codons can be read at the wobble codon (the 3rd codon)

Question 33

What does Aminoacyl-tRNA Synthetases do?

Answer 33

Attach amino acids to specific tRNA molecules

Question 34

Why must aminoacyl-tRNA synthetases be very specific?

Answer 34

As some amino acids have very similar structures

Question 35

What does proofreading require?

Answer 35

ATP

Question 36

What are the two sites in aminoacyl-tRNA sythetase?

Answer 36

• active site
• proofreading/editing site

Question 37

What happens at the editing site of aminoacyl-tRNA sythetases?

Answer 37

Incorrect amino acids that are similar to the correct one are recognised and broken down or changed

Question 38

What two subunits can ribosomes (70S) dissociate into?

Answer 38

Large (50S) and Small (30S)

Question 39

Which is more prevalent in ribosomes, proteins or RNA?

Answer 39

RNA is much more important - it is the functional core responsible for decoding of mRNA and protein formation. Proteins are on the outside and are for scaffolding

Question 40

What is a ribozyme and how is it different to an enzyme?

Answer 40

Both provide catalytic routes for reactions - however ribozymes and made up of RNA where as enzymes are made up of proteins

Question 41

What is the amino acid and codon used in initiation of translation?

Answer 41

Formyl-methionine, AUG

Question 42

What is the Shine-Dalgarno sequence?

Answer 42

An upstream purine-rich ribosome binding site

Question 43

What happens at the P-site during initiation of translation?

Answer 43

fMet-tRNA binds - all other aa-tRNA’s bind at the A site

Question 44

What are the role of initiation factors?

Answer 44

They help ensure correct order of assembly of the ribosome for transcription

Question 45

What is the role of a release factor in termination of translocation?

Answer 45

If STOP codon is read then a release factor binds to the A site and promotes hydrolysis of the terminal peptidyl-tRNA bond, release of the polypeptide and dissociation of the 50S-30S complex