Lesson 11: Transcription Part II: Specialities of Eukaryotes

Question 1

What happens when mRNA is transcribed in a prokaryotic cell?

Answer 1

In prokaryotes, mRNA is generated in the cytoplasm and ready to be translated immediately

Question 2

What happens when mRNA is transcribed in a eukaryotic cell?

Answer 2

In eukaryotes, the RNA transcript (pre-mRNA) is generated in the nucleus and needs to be processed and become a mature mRNA before being translated in the ribosome.

Question 3

What mechanisms could a cell use to determine if an mRNA was complete?

Answer 3

The cell can monitor the beginning, middle, and end of a transcript

Question 4

What is the RNA transcript called directly after transcription?

Answer 4

Pre-mRNA
- The pre-mRNA must be processed into a mature mRNA

Question 5

What are the different parts in a final mRNA transcript?

Answer 5

1) 5’ cap - eukaryote-specific
2) 5’ untranslated region (UTR)
3) Coding sequence (after introns were removed)
4) 3’ untranslated region (UTR)
5) Poly-A tail - eukaryote specific

Question 6

What is the purpose of an untranslated region in the final mRNA?

Answer 6

Is not used to build the protein but it has important regulatory information

Question 7

What is the purpose of the coding sequence in the final mRNA?

Answer 7

The part of the mRNA that gets converted into protein

Question 8

How does an eukaryotic cell monitor the beginning of the transcript?

Answer 8

By adding the 5’ cap

Question 9

What is a 5’ cap?

Answer 9

An atypical guanine added to the 5’ nucleotide of the RNA transcript by a 5’ to 5’ triphosphate linkage.

Question 10

How is the 5’ cap added to the pre-mRNA?

Answer 10

It is added by capping enzymes that associate with the synthesis of RNA polymerase

Question 11

What is the PURPOSE of a 5’ cap?

Answer 11

5’ cap Functions:
1) recruits proteins that facilitate nuclear exit
2) these proteins also protect the mRNA from being broken down
3) mark the transcript as a “mRNA” and lets the ribosome know to start reading it to make a protein
4) helps the ribosome attach to the mRNA by serving as a landing pad

Question 12

How does an eukaryotic cell monitor the end of the transcript?

Answer 12

By adding a poly-A tail

Question 13

What is a poly-A tail?

Answer 13

A string of adenine nucleotides that is added to the end of the transcript

Question 14

How is the poly-A tail added to the pre-mRNA?

Answer 14

Proteins that recognize a specific sequence cut the end of the transcript. Poly (A) polymerase then adds the poly-A tail.

Question 15

What is the PURPOSE of a poly-A tail?

Answer 15

Very similar function to the 5’ cap

1) recruits proteins that facilitate nuclear exit
2) these proteins also protect the mRNA from being broken down
3) mark the transcript as a mRNA

Question 16

How does an eukaryotic cell monitor the middle of the transcript?

Answer 16

By removing introns
- Note: Prokaryotes DO NOT have introns

Question 17

What is RNA splicing?

Answer 17

Introns are recognized and removed from the pre-mRNA and the exons are pasted together

Question 18

What are the steps of RNA splicing?

Answer 18

1) The 2’ OH group in an adenine nucleotide in the intron sequence (BRANCH-POINT adenine) attacks the 5’ splice site, breaking the sugar-phosphate backbone.
2) Then the splice site becomes covalently attached to the adenine
3) This frees an OH group on the 3’ end of an exon which then reacts with the 5’ end of the next exon joining the pieces together
4) The intron is released as a LARIAT structure

Question 19

Why do eukaryotic cells have introns?

Answer 19

• Introns allow for DNA to have a buffer region. If a mutation occurs it may not have an affect on our coding sequence
• Allows for alternative splicing which increases the diversity of the proteins a cell can make
• Important for protein evolution

Question 20

Can the number and length of introns vary?

Answer 20

Yes!

The number and length of introns can vary dramatically between genes
ex: the human beta-globin gene vs. the human factor VIII gene

Question 21

How does the eukaryotic cell know where to cut?

Answer 21

There are special sequences in the pre-mRNA that tells the cell there is a junction (intron-exon border) between an exon and intron
- Note: Must be precise, mistakes can shift reading frame

Question 22

What removes the introns?

Answer 22

Spliceosome - protein-RNA complex
- Note: snRNA is the specific RNA that is in the spliceosome. snRNAs are an example of functional RNAs that do not get translated to protein. Therefore, the spliceosome has many subunits of snRNPs (snRNA + protein)

Question 23

How does the spliceosome remove the introns?

Answer 23

Using complementary base pairing, spliceosomes position themselves at the intron/exon junction. The RNA in the spliceosome is what directs the location of binding and performs chemistry. Therefore a spliceosome is an example of a RIBOZYME.

Question 24

What is a ribozyme?

Answer 24

An RNA molecule or RNA-protein complex, in which the RNA provides catalytic activity
ex: ribosome and spliceosome

Question 25

How does the cell know RNA splicing is completed?

Answer 25

A protein is deposited to let the cell know that splicing has been completed

Question 26

What is alternative splicing?

Answer 26

Allows for MORE THAN ONE mRNA to be made from the same gene. These mRNAS are called ISOFORMS. - Note: These isoforms will retain the core elements of the protein but can change protein binding properties, alter cellular localization, enzymatic activity, protein stability, and post-translational modifications.

Question 27

What is the protein titin important for?

Answer 27

Titin is important for muscle properties and alternative splicing of mRNA that codes for titin is important during development

Question 28

How do changes in splicing during heart development alter properties of the protein titin?

Answer 28

When you are a fetus, all exons are used in the mRNA because you have low RBM20, resulting in a long, springy isoform, which leads to an elastic heart which is necessary for fetal hearts. When you are an adult, you have high levels of RBM20 which exclude some of the exons, resulting in a short, stiff isoform, which is important for proper heart function as an adult. NOTE: Adult patients who have a mutation and have higher fetal titin (long, springy titin) have a condition called dilated cardiomyopathy, which is when the heart is too elastic.

Question 29

How do complete mRNAs leave the nucleus?

Answer 29

Nucleus acts like a bouncer and does not allow the mRNA to leave the nucleus until its complete. Once it is complete, different proteins bind to the complete mRNA and allow it to leave the nucleus.

Question 30

How does the cell ensure all these processes happen properly?

Answer 30

Coordination

Question 31

How does coordination occur?

Answer 31

1) Kinases phosphorylate amino acids (serines and threonines) on the C-terminal Tail Domain of RNA polymerase II 2) This recruits capping, splicing, and polyadenylation factors, which makes sure all of the processes are happening correctly. NOTE: Also allows "promoter escape" of RNA polymerase II. Phosphorylating the C-terminal Tail Domain allows RNA polymerase to detach from the promoter allowing for RNA polymerase to synthesize the transcript in the elongation phase. 3) Coordination of RNA processing events occurs as the RNA polymerase moves along the DNA making the mRNA.

Question 32

How can a cell control how much protein is made by a protein coding gene?

Answer 32

In both eukaryotes and prokaryotes the amount of a protein in a cell depends on the rates of mRNA synthesis and RNA processing steps. Also, the rate of degradation of mRNA. - Note: Strong promoters make more RNA (so more protein)

Question 33

How do we turn off or degrade an mRNA?

Answer 33

1) A decapping enzyme can remove the 5' cap 2) An enzyme can remove the poly-A tail 3) mRNA can get recognized by exonucleases which destroys the RNA by breaking it down to ribonucleotides to be recycled.

Question 34

Are these mRNA processing mechanisms specific for eukaryotes?

Answer 34

Yes! In general, prokaryotic mRNAs are very short lived so they do not have as many mechanisms to control stability.