Chapter Five

Question 1

Is the genome unwound when it is transcribed?

Answer 1

No, only certain, specific sections are kept unwound

Question 2

Why, like DNA, can RNA only be copied from DNA in the 5’ to 3’ direction?

Answer 2

Because it needs to attach to the 3’ hydroxyl

Question 3

What are the five types of RNA that we discussed?

Answer 3

1. mRNA
2. tRNA
3. rRNA
4. snRNA
5. miRNA

Question 4

What is mRNA?

Answer 4

messenger RNA “genes”
it is the message for the order of amino acids

includes 3000 ish base pairs (1500-8000)

Question 5

What is tRNA?

Answer 5

transfer RNA
it transfers or escorts amino acids to the ribosome complex to convert mRNA to the chain of amino acids

91 bases long

they fold in on themselves making clover like structures.

Question 6

What is rRNA?

Answer 6

Ribosomal RNA
RNA structurally important for the ribosomal complex

it connects the protein complexes of the ribosome

Question 7

What is snRNA?

Answer 7

small nuclear RNA
it interacts with proteins to form snURP’s, which play a role in RNA processing

usually a few hundred bases long

Question 8

What is miRNA?

Answer 8

micro-interference RNA

21 bases long (used in gene regulation)

Question 9

What stage of the cell cycle does transcription take place?

Answer 9

G2 (cell growth)

Question 10

What is the first step of DNA transcription?

Answer 10

Initiation

Question 11

How is it recognized that a certain bit of DNA needs to be transcripted?

Answer 11

There exists sequences adjacent to the DNA that act as promoter sites.

Question 12

What is the core promoter?

Answer 12

It is a region of DNA adjacent to the gene, that encompasses the lnr and TATAAA box

Question 13

What is the TATAAAA box?

Answer 13

It is a region of the DNA upstream from the gene within the core promoter and is about 25-30 base pairs away from the TSS

Question 14

What is the CATT box?

Answer 14

It is a region of DNA that exists around 75 base pairs upstream from the gene that helps in promotion of transcription

If there is a mutation within this region, only 4/10 times will the gene that lies further downstream be transcribed

Question 15

What is the GC box?

Answer 15

It is a region of DNA that exists around 90 base pairs upstream from the gene that also helps as a promoter.

Question 16

Explain the initiation step in transcription in as much detail as possible.

Answer 16

TFII D which is a protein involved in initiation is bouncing around within the nucleus and binds to the TATAAAA box. This flexes the protein and allows other proteins that assist in initiation (TFII A & B) to come along and bind to TFII D. This is the start of the assembly of the pre-initiation complex.

The entire point of the these proteins, is to orient themselves so that TFII F attached to RNA Polymerase can come along and add to the complex. This is now the minimal transcription complex.

Finally, TFII E and H come along and bind tightly to the complex to stabilize the RNA Polymerase, completing the pre-initiation complex.

Question 17

Describe the elongation step in transcription in detail.

Answer 17

The RNA polymerase II has its own helicase activity. So RNA poly II is able to embed in between the DNA strands and move along. There is no need to SSBP to hold it open. As RNA Polymerase II is moving down and copying, it has no proofreading activity. The RNA Polymerase II makes these chains of RNA that drag behind it as nucleotides are added. The ten most recently made RNA nucleotides are still adhering to the single strand of DNA being copied, and as nucleotides are added, the mRNA breaks off form the pressure of the turns.

This is because RNA is bigger and cannot continue to adhere to the DNA because DNA is smaller and has alpha helix turns that RNA cannot follow bc RNA cannot fold that small.

This tail continues to build. Once the RNA Polymerase II reaches the end of a gene at a stop codon, the RNA stops copying

Question 18

What things can RNA Polymerase carry out?

Answer 18

1. Helicase activity (it unzips)
2. 5’ to 3’ polymerase activity (it adds)
3. Like DNA primase, it does not require 3’-OH to start a chain of RNA

Question 19

What does RNA Polymerase not carry out?

Answer 19

1. It has no 3’ to 5’ exonuclease activity (proofreading)
2. It has no 5’ to 3’ exonuclease activity

(we aren’t running into the back of any DNA)

Question 20

Why is 3’-5’ exonuclease activity not important for transcription?

Why is it important for DNA replication?

Answer 20

Proofreading is not so important for DNA transcription because RNA is mass produced, and it is very unlikely that the same mistake will exist for all the produced RNA. If some have mistakes, its all good because they will get degraded.

DNA needs to be copied perfectly, or as close as we can get, because it is passed onto progeny, and without exonuclease activity, many mutations could occur that have negative effects for the cell.

Question 21

Can initiation and elongation happen simulataneously?

Answer 21

Yes!

Question 22

What are the two ways that transcription can be terminated?

Answer 22

Rho dependent process (termination proteins)

Rho independent process

Question 23

Explain how a gene that is Rho dependent terminates.

Answer 23

As RNA polymerase II approaches the termination site on the DNA, the Rho protein binds to a specific site on the newly synthesized RNA, known as the rut site past the stop codon. Rho then travels along the RNA toward RNA polymerase and when it catches up, there is no room for RNA polymerase II and it dissociates from the DNA releasing the RNA transcript.

Question 24

How does Rho independent termination work?

Answer 24

At the end of the RNA transcript, there exists, first, regions with high GC bases going (GCGCGCGC) and second, a region with high TU bases.

As RNA polymerase synthesizes the high GC region, it folds back on itself forming a hair-pin structure due to hydrogen bonding.

Because the only thing left after the high GC bases are TU, and these are weaker, the strand just disassociates from the DNA template given the pull of the hair-pin.

Question 25

How fast is RNA Polymerase?

Answer 25

80 bases per second.

Question 26

Generally speaking, what are the RNA processing steps?

Answer 26

1. 5’ capping
2. Intron Removal
3. Polyadenylation

Question 27

What needs to happen before the RNA can leave the nucleus? WHY?

Answer 27

If any of the three mRNA processing steps—5’ capping, polyadenylation, or splicing—fail, the RNA will not undergo the necessary structural changes. Because the enzymes remain attached, the RNA may become too large to pass through the nuclear pores, preventing it from leaving the nucleus.

Question 28

Describe what 5’ capping is and what it functions to do?

Answer 28

It is the addition of an upside down Guanine triphosphate on the 5’ end of the RNA strand, and then it become methylated (addition of CH3 to the upside down G) which allows for stability against degradation and will help line up mRNA on a ribosome for translation)

It acts like a shoe string cover and prevents degradation as well aiding in transcription setup.

Question 29

What is intron removal aka exon splicing?

How does it work?

Answer 29

Intron removal is when non-coding sequences (introns) are cut out of the transcribed mRNA, which usually has more introns than exons. Small nuclear ribonucleoproteins (snRNPs), made up of small nuclear RNA (snRNA) and proteins, are essential for this process. Each intron has a GU sequence at the 5’ splice site, an A branch site, and ends with an AG sequence at the 3’ splice site. During splicing, U1 snRNP binds to the GU site at the 5’ end of the intron, and U2 snRNP binds to the A branch site, causing the RNA to bend and bringing the splice sites closer together. After that, U4, U5, and U6 work together to stabilize the spliceosome and catalyze the splicing reaction. Together, these proteins make sure introns are accurately removed and exons are joined. Ribozymes embedded within the spliceosome are responsible for the enzymatic activity involved in splicing.

Question 30

What is alternative splicing?

Answer 30

You can get multiple proteins from a single gene because the splicing is a different combination.