RNA Synthesis

Question 1

Describe an RNA molecule

Answer 1

1. RNA is a single stranded polymer of nucleotides where the pentose sugar is always ribose and the organic bases are adenine, guanine, cytosine, and uracil.

Question 2

How does the RNA differ from DNA(4)

Answer 2

● RNA has ribose nucleotides not deoxyribose nucleotides ● RNA has uracil instead of thymine ● RNA is single stranded not double like DNA ● RNA is shorter than DNA

Question 3

Describe the structure of mRNA

Answer 3

It is a long single-stranded molecule of up to thousands of nucleotides which can contain one gene only

It is manufactured in the nucleus and is the mirror copy of part of one strand of the DNA helix.

It makes up less than 5% of the total cellular RNA.

It is easily and quickly broken down sometimes existing only for minutes.

Question 4

What is mRNA?

Answer 4

“Messenger RNA carries the ‘message’ that codes for a particular protein from the cell nucleus to the cytoplasm where proteins are synthesized.”

Question 5

Describe the structure of tRNA

Answer 5

tRNA is a small molecule which is 80 nucleotides long which is also single-stranded.

2. It makes up 10- 15% of the cells RNA and all types are fundamentally similar
3. It forms a clover leaf shape with one end of the chain having the base sequence ACC where the amino acid binds
4. On the middle loop there is a triplet nucleotide sequence called the anticodon.
5. There are 64 different types of tRNA molecules each with a different anticodon sequence complementary to the 64 different codons.

Question 6

How are amino acids attached to their tRNA molecules?

Answer 6

By specific aminoacyl tRNA synthase enzymes.

Question 7

Describe the structure of rRNAs

Answer 7

“rRNA is a complex molecule made up of both double and single helices.

It is made in the DNA of the nucleus it is found in the cytoplasm

Makes up more than half the mass of the ribosomes.

They are made of two sub-units, small and large and are assembled in the nucleolus of the nucleus and exported into the cytoplasm.

rRNA is coded for by numerous genes in many different chromosomes.

Question 8

What are the five requirements for transcription to occur? (6)

Answer 8

1. DNA template
2. RNA polymerase II- transcribing protein coding genes
3. Buffer
4. rNTPs- the building blocks of RNA
5. Transcription factors- used for recruitment of RNA polymerase II because RNA polymerase II in eukaryotes does not recognize and bind to promoter regions unlike in prokaryotes so needs a TF.
6. Promotor regions- acts as a ‘bookmark’ so RNA polymerase knows where to transcribe”

Question 9

At what speed does transcription occur?

Answer 9

1.25-1.75kb per min

Question 10

What is the role of transcription factors?

Answer 10

· Help to position the RNA polymerase correctly at the promoter,

· Aid in pulling apart the two strands of DNA to allow transcription to begin,

· Release RNA polymerase from the promoter into the elongation mode once transcription has begun.

Question 11

How long is the TATA box?

Answer 11

25 nucleotides

Question 12

What is the CAAT box?

Answer 12

CAAT box with a consensus sequence of GGNCAATCT which is found 75 nucleotides upstream of the gene

signals the binding site for the RNA transcription factor

Question 13

Differences between CAAT box and TATA box

Answer 13

“TATA box is a conserved nucleotide region found about 25-30 base pairs upstream to the transcription initiation site but CAAT box is a conserved region of nucleotides found about 75-80 base pairs upstream to the transcription initiation site.

TATA box provides a binding site for TBP and transcription factors and participates in transcription regulation while the CAAT box signals the binding site for the RNA transcription factor.

Question 14

What are enhancers?(3)

Answer 14

“1. Not found in prokaryotic cells but it is a sequence on which a protein binds and it forms a complex which promotes the process of transcription and increases the efficiency of RNA polymerase.

2. They can be found upstream or downstream with respect to the eukaryotic cell.
3. They are usually 1000 nucleotides away from the gene that is being transcribed.

Question 15

What does the TFIID consist of?

Answer 15

TFIID is made of multiple sub-units and one of the subunits is called TBP (TATA binding protein)

TFIID also consists of around 10-12 other polypeptides called TBP associated factors or TAF for short

Question 16

What is the role of TFIIB and TFIIH?

Answer 16

“TFIIB is responsible for linking RNA Polymerase to the whole complex of TBP and TFIID.

TFIIH then uses ATP to pry apart the DNA which is called a cistron at the transcription start point, allowing transcription to begin.

TFIIH contains helicases which allows it to gain access to the template

Question 17

Describe the process of initiation in RNA synthesis?

Answer 17

→TFIID binds to the promoter region (TBP is a subunit of TFIID tata binding protein)
→TFIIB binds to the TBP and it links RNA polymerase to the TFIID
→WHEN RNA polymerase binds to the TFIIB it is already associated with TFIIF
→TFIIE and TFIIH associate with RNA polymerase

.TFIIH then uses ATP to pry apart the DNA which is called a cistron at the transcription start point TFIIH contains helicases which allows it to gain access to the template

6. TFIIH also phosphorylates RNA polymerase II, changing its conformation so that the polymerase is released from the general factors and can begin the elongation

Question 18

Describe the elogation process(2)

Answer 18

1. RNA polymerase ““walks”” along one strand of DNA, known as the template strand, in the 3’ to 5’ direction.
2. For each nucleotide in the template, RNA polymerase adds a matching (complementary) RNA nucleotide to the 3’ end of the RNA strand.

Question 19

Describe the process of the 7-methylguanine cap

Answer 19

· As transcription occurs, the 5’ end of the transcript contains a free triphosphate which can be digested.

After 25 nucleotides of RNA are synthesized the 5’, a guanine is added to the 5’ end and an enzyme called methyl transferase methylates (also called guanyl transferase) the guanine at position 7 to give rise to 7 methylguanine cap.

· The cap is formed through a 5’-5’ linkage between the two substrates

· The cap is co-transcriptional modification and, in the nucleus

the cap binds a protein complex called CBC (cap-binding complex), helps the RNA to be properly processed and exported

Question 20

What direction does RNA polymerase lay the nucleotides?

Answer 20

RNA polymerase reads a DNA template in the 3’ to 5’ direction to synthesize RNA in the 5’ to 3’ direction.

Transcription can occur in either direction, but not in either direction from a single promoter

Question 21

Describe the process of polyadenylation

Answer 21

“· Near the stop codon, there is a sequence AAUAAA which is the polyadenylation signal or termination signal, after the stop codon.

The 3’ end of the transcript is cleaved to free a 3’ hydroxyl by CPSF (cleavage and polyadenylation specificity factor). Then an enzyme called poly-A polymerase adds a chain of adenine nucleotides to the RNA

. This adds a poly-A tail that is between 100 and 250 residues long.”

Question 22

Why is polyadenylation important?

Answer 22

· The poly-A tail makes the RNA molecule more stable and prevents its degradation.

allows the mature messenger RNA molecule to be exported from the nucleus and translated into a protein by ribosomes in the cytoplasm.

· The longer the poly A tail, the more stable the RNA is.

Question 23

What are the base letters at the intron/extron junction?

Answer 23

The G/GU at the 5 ′ end of the intron ( the 5 ′ splice site ),

the AG/G at the 3 ′ end of the intron ( the 3 ′ splice site

Question 24

Describe the structure of a spliceosome

Answer 24

· Each spliceosome is made up of 5 small nuclear RNAs and a range of associated protein factors,

· when the small RNAs are combined with the protein factors they make RNA-protein complexes called snRNPS (small nuclear ribonucleoproteins). · The snRNAs that make up the major spliceosome are U1, U2, U4,U5,U6 which are rich in uridine.

. snRNAs are transcribed by RNA polymerase II or RNA polymerase III.

Question 25

Describe the process of splicing

Answer 25

→Splicing is the removal of introns and the joining of exons.

→There is cleavage at the 5’ site by a splicosome.

→formation of a lariat-like intermediate (GU joining to A at the branch point, making an AGU)

→Then, ligation joining together of exons occurs.

Question 26

Why is alternative splicing important?

Answer 26

· Allows the production of more than one mRNA, and therefore more than one protein product, from a single gene.

Question 27

In some cases, why does alternative splicing occur?

Answer 27

“1. Intron ambiguity- the standard splicing mechanism for removing intron sequences is unable to distinguish clearly between two or more alternative pairings of 5’ and 3’ splice sites so that different choices are made.

Question 28

What are some of the checks in place to ensure the right mRNA leaves the nucleus?

Answer 28

· Cap- binding complex, which recognises the cap

· TREX = transcription-coupled export complex- · EJC = exon-junction complex- recognises successful slicing events

· All these proteins present indicate mRNA is properly processed.

Question 29

What are the differences between DNA synthesis and RNA synthesis?

Answer 29

Enzymes Choice of Substrate Products Choice of which part of Template to use

Question 30

What are signal sequences?

Answer 30

· Signal sequences are amino acids at the N-terminal end of the protein that are recognised by enzyme systems within the cell that transport the protein to the correct destination

Question 31

Explain the role of the COP proteins

Answer 31

A protein complex that coats vesicles transporting proteins between Golgi compartments and the formation of buds

Question 32

Recall the different types of RNA polymerase and the genes transcribed

Answer 32

RNA polymerase I- most ribosomal RNA

RNA polymerase II- protein coding, microRNA, non-coding RNA

RNA polyemrase III- tRNA, 5s rRNA and other small RNAs

Question 33

Name different types of alternative splicing

Answer 33

1. Cassette exon eg. troponin
2. mutually exclusive exons
3. intron retension
4. alternative 5’SS eg tropomyosin
5. alternative 3’ SS
6. alternative promotors eg myosin
7. alternatve polyadenation eg. ubiquitiously expressed genes

Question 34

Importance of export proteins:

Answer 34

→Helps cell distinguish between rare mature mRNA and RNA processing debris.