Lec 3-4 Restriction endonucleases

Question 1

Restriction Endonucleases Roles

Answer 1

enzymes which:
Cleave a specific DNA sequence

Protect bacteria from phage infection digesting phage DNA after infection
cellular

DNA is protected by methylases blocking restriction an enzyme activity

Question 2

Restriction site

Answer 2

specific sequence recognized by restriction enzymes (4-6 bps) (could be 8)

Question 3

Fusion tag characteristics (5)

Answer 3

*Improved solubility (S)

*Improved detection (D)

*Improved purification (P)

*Localization (L)

*Improved Expression (E)

Question 4

Improved solubility (S)

Answer 4

• Fusion of the N-terminus of the target protein to the C-terminus of a soluble fusion partner often improves the solubility of the target protein.

Question 5

*Improved detection (D)

Answer 5

• Fusion of the target protein to either terminus of a short peptide (epitope tag) or protein which is recognized by an antibody (Western blot analysis) or by biophysical methods (e.g. GFP by fluorescence) facilitates the
  detection of the resulting protein during expression or purification.

Question 6

*Improved purification (P)

Answer 6

• Simple purification schemes have been developed for proteins used at either terminus which bind specifically to affinity resins.

Question 7

*Localization (L)

Answer 7

• Tag, usually located on N-terminus of the target protein, which acts as address for sending protein to a specific cellular compartment.

Question 8

*Improved Expression (E)

Answer 8

• Fusion of the N-terminus of the target protein to the C-terminus of a highly expressed fusion
  partner results in high level expression of the target protein.

Question 9

Start codon

Answer 9

Start codon
The initiation codon in E. coli is most commonly AUG (met). About 8%
of start sites use GUG (val), whereas UUG (leu) and AUU (Ile) are rare initiators present in autogenously regulated genes.

Question 10

Rare codons

Answer 10

Rare codons
The frequencies with which the different codons appear in genes in E. coli are different from those in genes derived from other organisms. The amount of specific tRNAs is also reflected by the frequency of the codon, meaning that a tRNA which
recognizes a rarely used codon is present in low amounts. Therefore, various genes that contain codons which are rare in E.
coli may be inefficiently expressed by this organism. Rare codons can cause premature termintion of the synthesized protein
or misincorporation of amino acids. Clusters of rare codons stand a higher chance to create translation errors and reduce
the expression level. This problem can be solved either by exchanging codons in the target gene for codons which are more
frequently used in E. coli, or, alternatively, by co-production of the rare tRNAs.

Question 11

Stop codons

Answer 11

Stop codon
Termination of mRNA translation.
UAA, UAG and UGA.

Question 12

How to improve translational termination efficiency

Answer 12

E. coli displays a strong bias towards the UAA codon

Translational termination
efficiency is further improved by the prolonged UAAU sequence. Alternatively, two consecutive stop codons may ensure
termination.

Question 13

RBS

Answer 13

Ribosome Binding Site (RBS) is an RNA sequence found in mRNA to which ribosomes can bind and initiate translation.

Translation initiation in bacteria almost always requires both an RBS sequence and a start codon.

• usually 6 nucleotides upstream of a start codon in an mRNA.

The ribosomal holoenzyme binds to both the RBS and the start codon.

Question 14

Kozak sequence

Answer 14

Yeast RBSs, more often known as Kozak sequences, are designed to be recognized by the
yeast ribosome. As a eukaryotic translation signal, the sequence of yeast RBSs are distinct
from prokaryotic RBSs.

Question 15

Shuttle vectors

Answer 15

– contain origins of replication for
different species – plasmid replicates in both species