RNA Synthesis and Processing

Question 1

Transcription

Answer 1

DNA –> RNA

3 Steps:

1) Initiation
2) Elongation
3) Termination

Transcription, unlike replication, does not require a primer. It is also not very accurate.

Question 2

Translation

Answer 2

RNA –> Protein

Question 3

Intron

Answer 3

non-coding region of the DNA. Introns do not contain genes.

Question 4

Exons

Answer 4

Coding regions of the DNA that contain genes

Question 5

RNA Splicing

Answer 5

The process of removing introns

Question 6

Promoter

Answer 6

A region of the gene that initiates transcription. The TATA Box is a promoter. Some promoters can be upstream of the transcriptional start site, some downstream and some at it. Its job is to describe location and orientation of the gene it is going to transcribe. This is why it is has to be asymmetrical! If it was symmetrical, polymerase wouldn’t know which way to go.

Question 7

mRNA

Answer 7

messenger RNA. It codes for the protein

Question 8

rRNA

Answer 8

ribosomal RNA. forms the basic structure of ribsomes

Question 9

tRNA

Answer 9

transfer RNA. central to protein synthesis as adapter between mRNA and amino acids.

Question 10

snRNA

Answer 10

small nuclear RNA. Involved in splicing.

Question 11

snoRNA

Answer 11

small nucleolar RNA. Helps to chemically modify rRNAs

Question 12

miRNA

Answer 12

microRNA. regulate gene expression

Question 13

siRNA

Answer 13

small interfering RNA. turn off gene expression

Question 14

RNA Polymerase I

Answer 14

synthesizes rRNA. Requires a specific termination factor.

Question 15

RNA Polymerase II

Answer 15

Synthesizes mRNA, snoRNA, miRNA, siRNA and most snRNA. Terminates after the Poly-A tail (downstream of the poly-A tail)

Question 16

RNA Polymerase III

Answer 16

Synthesizes tRNA, 5S rRNA, some snRNA, and other small RNAs. Terminates after a series of U’s

Question 17

Discuss the relative abundance of each RNA type.

Answer 17

rRNA is the most abundant. Then tRNA and finally mRNA is the least abundant because it is rapidly degraded.

Question 18

Describe the function of a promoter in gene transcription

Answer 18

A promoter initiates gene transcription

Question 19

Upstream

Answer 19

denotes the 5’ end or to the left of the gene. Assigned negative numbers

Question 20

Downstream

Answer 20

denotes the 3’ end or to the right. Assigned positive numbers

Question 21

TFIIH

Answer 21

Has helicase activity and begins to unwind the DNA. It also has kinase activity and phosphorylates the c-terminal tail of the RNA polymerase II. This allows the polymerase to loosen its grip and get transcription.

Question 22

TFIID

Answer 22

Contain the TBP (TATA Binding Protein). It then recruits other transcription factors (TFIIA and TFIIB) which will then cause the recruiting of the other transcription factors and the RNA polymerase II.

Question 23

TBP

Answer 23

TATA Binding Protein. It binds to the TATA box in a sequence specific manner and distorts the DNA. This marks the location of an active promoter and allows for the assembly of RNA pol II and other TFIIs to form the transcription initiation complex. TBP binds to the minor groove and forms a saddle-like structure for other transcription factors to sit on top of. It bends the DNA towards the major groove, widening the minor groove.

Question 24

TAFs

Answer 24

TATA Binding Protein Associated Factors. If there is no TATA box they can associate with the DNA in a specific manner and force the TBP to interact with the DNA.

Question 25

Pre-mRNA

Answer 25

The pre-mRNA actually doesn’t exist. You can’t actually isolate it because it is being modified as it is being transcribed. The factors that process the mRNA are associated with the c-terminal tail (NOT THE N-TERMINAL LIKE HISTONES) of the polymerase. It would hypothetically contain introns and exons and not have a cap or a tail.

Question 26

5’-Cap

Answer 26

it is a 7-methylguanine cap that is attached in a 5’ to 5’ manner. It protects the 5’ end from degradation and it put on when ser5 of the c-terminal tail is phosphorylated and recruits the capping protein. Occurs by phosphatase removing a phosphate from the 5’ end, then Guanyl Transferase adding a Guanine to it. Then Guanine is methylated by methyl transferase.

Question 27

What are the primary functions of the 5’ Cap and the Poly-A Tail?

Answer 27

1) Allows the protein synthesis machinery to determine if the mRNA is intact
2) It distinguished the mRNA from other RNAs
3) It protects the mRNA from degradation
4) It facilitates the mRNA being moved out of the nucleus
5) It helps to maintain the mRNA stability for translation

Question 28

What are the Polyadenylation and Termination Steps?

Answer 28

1) CPSF (cleavage and polyadenylation specificity factor) recognizes the AAUAAA sequence and binds to it.
2) CSTF (cleavage stimulation factor) binds to the GU-rich or U-rich region. There is then cleavage at the CA.
3) Poly-A Binding Proteins are then recruited and add the poly-A tail with the Poly-A polymerase.

This is much more complicated than in bacteria.

Question 29

Splicing Singals to Know

Answer 29

5’ Splice Donor Site: “GU”
3’ Splice Donor Site: “AG”
Branch Point: “A”

SR Proteins mark the exon, intron junctions.

Question 30

Splicing Reaction

Answer 30

The splicing reaction is catalyzed by snRNPs (small nuclear RNA-protein complex) called the spliceosome. It is a double transesterification reaction, one at each splice site.

Steps:
1) U1 snRNP bind to the 5’ splice site . BBP (branch-point binding protein) and U2AF (U2 auxiliary factor) recognize the branch-point site, the “A”.

2) U2 snRNP replaces the BBP and U2AF, binding to the branch point A and forms a catalytic center.
3) U4/U6 and U5 “triple” snRNP comes in and forms the lariat that will be cleaved. The U4 masks the catalytic activity of the U6 until it is released.
4) U1 and U4 leave, cleaving of the 5’ splice site now takes place because U6 is active without U4. U6 cleaves the 3’ splice site as well to produce the lariat.
5) EJCs (exon junction complexes) bind to the exons to stabilize them.

Question 31

snRNPs

Answer 31

they are snRNAs that are associated with proteins. They guide splicing by base-pairing with complementary sequences in the RNA. ALL RECOGNIZING THE mRNA BASED ON BASE PAIRING.

Question 32

AT-AC Splicing

Answer 32

Another form of splicing that uses alternate splicing sites instead of the GU and AG site.

Question 33

tRNA Splicing

Answer 33

The mechanism for splicing tRNA is unique because it is a “cut-and-paste” mechanism and does NOT involve a lariat.

Question 34

Splicing Mutations

Answer 34

If splicing is not done correctly, it can lead to mutations. Beta-Thalassemia is an example in which a 5’ splice site is destroyed. When this occurs, due to there already being aa 3’ splice site and so there aren’t two 5’ splice sites, a new, cryptic 5’ splice site is introduced which then alters the splicing and deletes exon regions or inserts intron regions. the 5’ and 3’ splice sites occur in PAIRS and when one is removed a new cryptic one will be introduced. A similar case is when a 5’ splice site is generated by a mutation. Now a new 3’ cryptic splice site is introduced to get the other pair.

Question 35

mRNA Export

Answer 35

Some protein remain bound to the mRNA when it is transported out of the nucleus and some do not.

Question 36

Synthesis of rRNA

Answer 36

There are two important promoter elements:

1) upstream control element (UBF or upstream binding factor binds here)
2) Core element (core promoter)

They need to be properly distanced to avoid steric hinderance. There is not a TATA box in this case but TBP still binds, using TAFs to force it on. rRNA is synthesized as a single large 45S precursor that will make 3 of the 4 rRNAs that make up ribosomes (5.8S, 18S, and 28S). The 45S will have half of it removed and the other half will undergo over 100 methylations or 2’-OH and 100 isomerizations of uridine to pseudouridine (modifications are done by snoRNP). It is thought these modifications are necessary to form the final ribosome structure.

Question 37

What is the site of ribosome synthesis and assembly?

Answer 37

nucleolus

Question 38

tRNA and 5S rRNA synthesis

Answer 38

RNA polymerase III transcribes tRNA (and 5S rRNA). The A-box and C-box promoters are used to synthesize 5S rRNA and the A-box and B-box promoters are used to synthesize pre-tRNA. TBP is involved. The TFIIA is specific for 5S rRNA synthesis and binds to the C-box promoter. THE PROMOTERS ARE DOWNSTREAM OF THE TRANSCRIPTIONAL START SITE

Question 39

tRNA Structure

Answer 39

tRNA undergoes extensive modifications. Over 50 different modifications can occur and 1 of 10 nucleotides are modified. It is thought that these modifications are necessary for its folding. Such modifications can include:

1) Removal of a short intron in the anticodon loop (not all tRNAs have introns)
2) Removal of a short nucleotide sequence from the 5’ end of the pre-tRNA.
3) Replacement of 2 uridine nucleotides at the 3’ end with CCA
4) Modifications of specific bases in the tRNA.

It is an overall clover structure.