RNA synthesis

Question 1

How many chromosomes do humans have?

Answer 1

23 pairs of chromosomes
22 pairs of autosomes
1 pair of sex chromosomes

Question 2

Give the two definitions of a gene

Answer 2

1. Unit of heredity; contains instructions for making an organism’s phenotype
2. DNA segment containing instructions for making a particular product

Question 3

What is the promoter region of a gene?

Answer 3

The promoter region is a short base sequence which determines whether a gene is going to be turned off or on which is really important in terms of gene expression

Question 4

What are exons and intron?

Answer 4

Exons are expressed sequences in the DNA

Introns are sequences in the DNA that are spliced out

Question 5

Where are the untranslated regions?

Answer 5

At the beginning of the first exon and the end of the last exon

Question 6

What are the differences between transcription and translation?

Answer 6

Transcription is the synthesis of mRNA transcript from DNA
It occurs in the same ‘language’- nucleic acids
Translation is the protein production from mRNA transcript
It occurs in different ‘languages’- nucleic acid to protein

Question 7

What is the function of RNA polymerase II?

Answer 7

RNA polymerase II catalyses the synthesis of the phosphodiester backbone of the RNA strand

Question 8

What genes does each type of RNA polymerase transcribe?

Answer 8

RNA polymerase I transcribes mostly ribosomal RNA (rRNA)
RNA polymerase II ia protein coding and also transcribes microRNA (miRNA), non-coding RNA
RNA polymerase III Transfer RNA (tRNA), 5S rRNA and other small RNAs

Question 9

What does RNA synthesis require to begin?

Answer 9

DNA template 
RNA polymerase II
Ribonucleotides 
A buffer 
Transcription factors

Question 10

What are transcription factors and what do they do?

Answer 10

Transcription factors are proteins required to initiate or regulate transcription in eukaryotes
They assemble on promoter to position RNA pol II
Pull apart DNA helix and expose template strand

Question 11

How do the different transcription factors assemble (what does each one do)?

Answer 11

1. TATA box is recognised by the TATA-binding protein (TBP) a subunit of TFIID
2. TFIIA and TFIIB bind; TFIIA stabilises the complex
3. The other general transcription factors bind, E and H, bind
4. Then RNA Pol II assembles at the promoter , forming the transcription initiation complex
5. TFIIH pulls apart the DNA helix and phosphorylates RNA Pol II
6. Phosphorylated RNA Pol II is released from the complex and begins transcription

Question 12

Are UTRs transcribed and why?

Answer 12

UTRs are transcribed but not translated
5’ UTR for regulation of translation
3’ UTR for mRNA stability and miRNA binding

Question 13

What three processes does the primary transcript have to go through before leaving the nucleus?

Answer 13

Capping
Polyadenylation
Splicing

Question 14

Recall the capping process

Answer 14

Capping is done by the capping enzyme complex
The 5’-5’ triphosphate bridge is formed and methylated at the 7 position
(This is a co-transcriptional modification, meaning the capping is happening while the mRNA is still being transcribed)
The capping enzyme complex is recruited by the phosphorylated form of the RNA Pol II

Question 15

What is the purpose of capping?

Answer 15

The cap can act as a marker
It can stimulate splicing and is used for recognition of messenger RNA by the protein’s translation machinery in the cytoplasm

Question 16

Recall the process of polyadenylation

Answer 16

The cleavage signal is recognised and cleaved by specific endonucleases 
After cleavage we get the addition of a poly A tail by an enzyme called poly(A) polymerase 
The poly(A) polymerase adds about 200 As to the end of the RNA chain (this process uses ATP)

Question 17

Why do capping and polyadenylation occur?

Answer 17

1. Stability- the nucleus contains many nuclease enzymes ready to degrade RNA and the poly(A) tail prevents it from being degraded
2. Transport to cytoplasm- RNA must be fully formed before it leaves the nucleus and processes are in place to check that this has happened
3. Integrity prior to translation- the cell doesn’t want to waste energy resources translating an mRNA that is incomplete or incorrect

Question 18

Describe introns

Answer 18

Non-coding sequences within genes
Size: <100->700,000 nucleotides
Median: 1800 nucleotides (exon: 123 nucleotides)
Number/gene: 0 (e.g. histones) - 78 (dystrophin)
Median: 7-9

Question 19

Recall the process of splicing

Answer 19

All of this is done by a large enzyme complex called a spliceosome 
The GU (sequence) is recognised by an RNA within the spliceosome and the other proteins are binding
The pyrimidine rich region also gets binding of proteins 
The proteins undergo conformation change so that you get cleavage at the 5' end of the intron
The ends join up with the A site to form a lariat structure 
Then you have cleavage of the 3' end of the intron and finally ligation of the two exons together
The intron gets degraded in the nucleus

Question 20

What is the reason for introns existing?

Answer 20

Alternative splicing

This means there are many more proteins than there are genes so you can make a bunch of proteins from a couple of genes

Question 21

How does the mRNA leave the nucleus?

Answer 21

The CBC (cap-binding complex) recognises the cap
TREX (transcription-coupled export complex) joins up and then unbinds to make a large complex
The EJC recognises successful splicing events