Translation and Processing

Question 1

What is the start codon?

What are the 3 possible termination codons?

Answer 1

• Start = AUG
• Stop
  
  • UAA
  • UAG
  • UGA

Question 2

What is an open reading frame? What is their significance?

Answer 2

A series of 50+ codons without encountering a termination codon. A long ORF indicates a gene that codes for proteins.

Question 3

Prokaryotes

Why can ribosomes come along and translate as the mRNA is being transcribed? (i.e. they happen in tandem)?

Answer 3

Because prokaryotes don’t have a nucleus or membrane bound organelles –> all happens in cytoplasm

Question 4

Prokaryotes

What modifications are made to:

• rRNA
• mRNA
• tRNA

Answer 4

• Methylation, cleavage
• None
• Cleavage, base modification

Question 5

Prokaryotes

What is the shine-dalgarno sequence? How many are in one transcript?

Answer 5

Distinguishes ribosomal binding site. There can be many in one transcript. They help the ribosome distinguish separate proteins in the same transcript

Question 6

Eukaryotes

In Prokaryotes, multiple ribosomes can be translating different proteins from the same transcript. How is this process different in Eukaryotes?

Answer 6

In eukaryotes, translation cannot begin until the mRNA leaves the nucleus. When it does, one transcript can have multiple ribosomes acting upon it, but they will be translating the same message and thus making multiple copies of the same protein.

Question 7

Eukaryotes

What are the modifications that are made to:

• rRNA
• mRNA
• tRNA

Answer 7

• methyaltion, cleavage
• 5’ capping, 3’ poly A tail, splicing
• cleavage, base modification, splicing

Question 8

Eukaryotes

What is the C-terminal domain of RNA Pol II? How does it relate to the 5’ cap?

Answer 8

It is a tail of the polypeptide of RNA Pol II that is exposed and contains a number of repeats that can be phosphorylated to influence RNA processing.

Phosphorylation of sites on the CTD results in recruitment of the molecules needed to add the 5’ cap to mRNA

Question 9

Eukaryotes

• What is the 5’ cap?
• When is the 5’ cap added?
• How is addition of 5’ cap integrated to RNA Pol II function?

Answer 9

• 7 - methylguanosine, added to 5’ end of mRNA
• DURING TRANSCRIPTION, shortly after initiation, after about 20-30 bases have been added
• RNA Pol II pauses and will not continue until cap is added

Question 10

Eukaryotes

What is the purpose of the 5’ cap?

Answer 10

• Protects the mRNA from degradation by making it difficult for RNAses to bind.
• Helps the body recognize the mRNA as it’s own product and keep it from being targted for degradation
• Aids in transportation to cytoplasm

Question 11

Eukaryotes

Describe how the poly A tail is added.

Answer 11

RNA Pol II reaches the polyadenylation signal (AAUAAA) and adds bases beyond that signal until it begins adding a region of bases that are rich in Us. This is part of the 3’ UTR. The RNA Pol II will fall off the DNA/mRNA and the transcript will be cleaved near the poly U site. The poly A tail will be added to the 3’ end of the mRNA and the 3’ UTR that contains the poly - U site will be degraded.

Question 12

Eukaryotes

What are the functions of the poly A tail?

Answer 12

• Nuclear export
  
  • After the mRNA leaves the nucleus via pore, the poly A tail curls and associates with the 5’ cap, pulling the transcript into loop structure
• Stability
  
  • increases mRNA half life
• Translation
  
  • B/c Poly A tail associates with 5’ cap and creates a circular mRNA, this actually makes translation more efficient - the same ribosome can move along the mRNA and complete it’s translation and then be right back at the start of the mRNA to translate again.

Question 13

Eukaryotes

Is there only one poly A site in a gene?

Answer 13

No - there can be several. This can lead to multiple places for the transcript to be cleaved and thus multiple variants of the product from the same gene –> form of alternative splicing.

Question 14

What is a canonical intron?

Answer 14

One that has GU at the start of the intron and AG at the end of the intron

Question 15

Eukaryotes

Is splicing always 5’ to 3’?

Answer 15

No - mRNA can be spliced 5’ to 3’ or the reverse. Both are functionally equivalent, and splicing can occur in any order but it is usually a highly predictable and repeatable order

Question 16

Eukaryotes

Describe the mechanism of splicing.

Answer 16

Question 17

Eukaryotes

What effect does the length of the 5’ UTR and 3’ UTR have on the mRNA?

Answer 17

• Longer 5’ UTR = less efficient translation
• Longer 3’ UTR = longer half life

Question 18

Eukaryotes

The 5’ UTR can have various shapes / structure to it that affects translation. Describe 3 such examples of how 5’ UTR shape affects translation.

Answer 18

1. Sequence - A specific sequence present in 5’ UTR can bind regulatory proteins that affect translation
2. Seconday structure - the 5’ UTR can form hair pin loops such that the start codon is inside the loop and thus ribosomes cannot bind and translate mRNA unless the hairpin loop structure is separated
3. Riboswitches - Hairpin loops in 5’ UTR can interact with other small molecules that alter the shape of the 5’ UTR and move the position of the start codon

Question 19

Eukaryotes

Where are ribosomes assembled? Where are they located?

Answer 19

Assembled in nucleolus, located in cytosol

Question 20

Eukaryotes

Describe the process of elongation of protein.

Answer 20

Question 21

Eukaryotes

Describe the process of initiation of translation.

Answer 21

Question 22

Eukaryotes

Describe the process of termination of translation.

Answer 22

Question 23

Eukaryotes

Are all transcripts translated at the 5’ cap?

Answer 23

No - most mRNAs are translated from their 5’ cap but some have internal ribosome entry sites. These are usually linked to major biological changes, such as stress conditions, mitosis, differentiation.