Week 3: protein synthesis - transcription and translation

Question 1

Where do the different steps occur?

Answer 1

• Transcription occurs in the nucleus, in the cytoplasm

- Translation occurs in the cytoplasm, in ribosomes.

Question 2

What are ribosomes made of?

Answer 2

• Pure ribosomal RNA

Question 3

What is a tRNA?

Answer 3

• A transfer RNA carries amino acids, which are the monomers for a protein. It has one codon (anticodon) which binds to mRNA.

One part has anticodon attatched which binds to codon of mRNA and another part has the amino acid attached which will break off to join the polypeptide chain.

• Matches an amino acid to an mRNA codon via the tRNA anticodon

Question 4

mRNA vs tRNA?

Answer 4

mRNA has certain bases (in codons) to determine which tRNA molecules (in anticodons) can bind with amino acids and hence which proteins to can be made.

Question 5

What’s the central dogma of transcription and translation?

Answer 5

During transcription, the information in a DNA sequence (a gene) is copied into a complementary RNA sequence.

During translation, this RNA sequence is used to create the amino acid sequence of a polypeptide.

Question 6

What’s the role of transcription?

Answer 6

Transcription relays the information in DNA to mRNA

Question 7

What’s the role of translation?

Answer 7

Translation relays the info in mRNA to polypeptides.

Question 8

what’s pre-mRNA?

Answer 8

pre-mRNA is the direct product of transcription

Question 9

What are the steps of transcription?

Answer 9

During transcription there are several steps…

1) Initiation
2) Elongation
3) Termination
- Produces pre-mRNA

Aka

1) RNA polymerase binds to the thing and gets organsied
2) RNA starts making shit so there’s more so the chain gets longer hence elongation
3) RNA stops making the shit

Question 10

What needs to happen to pre-mRNA to make mature mRNA?

Answer 10

• add a 5’ cap
• trim the 3’ tail and add a poly-A tail
• remove introns

Question 11

What’s an intron?

Answer 11

An intron is a non-coding area of the pre-mRNA.

They only exist in eukaryotes.

Question 12

How do you remove an intron?

Answer 12

Introns are removed with enzymatic spliceosomes (snRNPs)

Question 13

What are the purposes of introns?

Answer 13

Introns are used in…

• Alternative RNA splicing
• Exon shuffling (increases distance)
• Occuring within gene sequences.

Question 14

How does pre-mRNA get to a ribosome?

Answer 14

Only once mature, mRNA will exit the nucleus via nuclear pores into cytosol.

How exactly? We don’t know! Tehe

Question 15

What’s the role of RNA polymerase?

Answer 15

During transcription of mRNA, RNA polymerase wraps around a DNA chain and opens up a “bubble” of unzipped DNA inside, is responsible for producing pre-mRNA as it moves along template from 3’ to 5’ direction until termination site.

Question 16

What are the steps in initiation of transcription?

Answer 16

RNA polymerase binds to the promotor. Inside this promotor there’s a TATA box to which a transcriptor factor protein binds and is recognised by the RNA polymerase. Here, after binding to the promotor it unzips the DNA chain to make a bubble, then starts moving down, closing up the gap behind it.

Transcription actually starts downstream of this promoter, but upsteam of start codon.

Question 17

What is the role of the transcriptor factor protein?

Answer 17

TF binds to TATA box inside promotor, upstream of start codon. RNA polymerase must recognise that this has happened to start transcription.

Transcription factors are proteins that help turn specific genes “on” or “off” by binding to nearby DNA. If there’s no transcription factor a gene can’t be expressed so regulates gene expression.

A transcription factor (TF) ) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA.

Question 18

What are the steps in elongation (in transcription)?

Answer 18

Elongation just describes RNA polymerase making mRNA once it’s attached.
RNA polymerase moves along template in 3’ to 5’ direction and produces RNA transcript by adding nucleotides (complementary to template) at 3’ end of growing template.

Question 19

What are the steps in termination of transcription?

Answer 19

When RNA polymerase reaches the termination site it is freed and so is the RNA transcript.

Question 20

What are stop codons?

Answer 20

Stop codons are termination signals for translation

Question 21

Where does transcription start?

Answer 21

For transcription to start, a transcriptor factor protein binds to a TATA box inside a promotor, and then RNA polymerase can recognise this and start downstream of promotor but upstream of start codon.

Question 22

What are the components needed to start transcription?

Answer 22

For transcription to occur the enzyme RNA polymerase and transcription factor proteins are both needed. Together, the transcription factors and RNA polymerase form a complex called the transcription initiation complex.

Question 23

Introns vs exons?

Answer 23

Introns = non-coding regions 
Exons = coding regions

Question 24

Why introns?

Answer 24

Introns serve to…

• Enable alternative RNA splicing
• Increase distance for exon shuffling in crossing over
• Occur within genes: increase rate of assembling new genes

Question 25

What happens to introns?

Answer 25

Introns are removed with enzymatic spliceosomes (snRNPs) through RNA splicing.

Question 26

Termination signal vs stop codon?

Answer 26

Termination signal: end of transcription

Stop codon: end of translation

Question 27

What’s a polyribosome?

Answer 27

A polyribosome is where several ribosomes read one mRNA strand at once.

Question 28

Where are ribosomes?

Answer 28

Ribosomes are in the ctyosol (free) or in the rough ER (bound).

Question 29

What’s the structure of a ribosome?

Answer 29

Ribosomes are made up of large and small subunits, with three sites; A, P, and E

Question 30

Function of a ribosome?

Answer 30

Ribosomes hold mRNA and charged tRNAs in correct positions to allow polypeptide assembly.

Ultimately allows RNA translation into proteins

Question 31

What’s the significance of the tRNA wobble?

Answer 31

the tRNA wobble allows the same tRNA anticodon to recognise multiple codons for the amino acid it carries..

e. g. means AAG can bond to both UUC and UUU (UUU an “atypical base pair”).
- Allows redundancy in the base

Question 32

What is the purpose of transfer RNA (tRNA)?

Answer 32

tRNA selects amino acids needed to build protein coded for by the gene being read in mRNA.

Each tRNA molecule binds to a specific amino acid and to a specific mRNA codon so the mRNA codon translates to tRNA anticodon joined to specific amino acid bonding site…

Question 33

What are the steps of the binding process of amino acids to a transfer RNA?

aka “charging” the tRNA with an amino acid

Answer 33

1) Amino acid binds to the enzyme aminoacyl-tRNA synthetase
2) tRNA joins complex and forms high-energy bond to amino acid (within structure) (ATP-> AMP)
3) tRNA and amino acid exit structure as “activated” amino acid or aminoacyl tRNA.

Question 33

What are the steps of the binding process of amino acids to a transfer RNA?

aka “charging” the tRNA with an amino acid

Answer 33

1) Amino acid binds to the enzyme aminoacyl-tRNA synthetase
2) tRNA joins complex and forms high-energy bond to amino acid (within structure) (ATP-> AMP)
3) tRNA and amino acid exit structure as “activated” amino acid or aminoacyl tRNA.

Question 34

What’s the structure of a ribosome?

Answer 34

A ribosome is comprised of a small and a large subunit. The large sub-unit contains the A, P, and E sites and is where the mRNA is read.

The A site is at the 3’ end and the E site is at the 5’ end, with ribosomes reading the mRNA in the 5’ to 3’ direction (vs transcription where DNA template is read 3’ to 5’ end.

Question 35

What are the three sites of a ribosome?

Answer 35

The three sites of a ribosome are the A, P, and E sites.

A: aminoacyl-tRNA site.
- Where charged tRNA anticodon binds to mRNA codon

P: peptidyl-tRNA site
- Where tRNA adds its amino acid to polypeptide chain

E: exit site
- Where tRNA is released to return to aminoacyl-tRNA synthetase to pick up another amino acid and repeat the process.

Question 36

What happens in the initiation of translation?

Answer 36

During the initiation phase of translation the ribosome is assembled from the small and large sub-units around a strand of mRNA.

Small sub-unit binds to mRNA upstream of start codon and shuffles down until start codon (AUG) is in the P site.

The corresponding (methionine-charged) tRNA to the start codon (UAC) binds to the start codon (if there are initiation factors) and this creates the initiation complex. Note, because non-AUG start codons are v. rare in eukaryotes most peptide chains start with methionine because it is the amino acid associated with the UAC anticodon to AUG.

The large ribosomal subunit then binds to the initiation complex

Question 37

What are the two reactions catalysed by the large ribosomal subunit in the elongation cycle of translation?

Answer 37

The large ribosomal subunit catalyses two reactions using the enzyme peptidyl transferase.

• It breaks the bond between the tRNA and the amino acid its carrying
• It forms a bond between the amino acid and the polypeptide chain its joining (called a peptide bond).

Question 38

What happens in the elongation cycle of translation?

Answer 38

In the elongation cycle of translation essentially the large ribosomal subunit snaps the amino acids off the tRNA at the P site and passes it back to the tRNA at the A site which then effectively shuffles down. The empty tRNA is then disconnected at the E site. This repeats as new tRNAs are brought in and bind using the anticodon to codon and move through the A, P, and E sites and as empty ones are passed to the E site and ejected and returned to get a new amino acid. Large ribosomal subunit catalyses breaking tRNA-amino bond and forming peptide bond to grow peptide chain, using the enzyme peptidyl transferase. Continues until a termination codon is reached.

Growing peptide chain on the P site shuffles back to the A site and then this tRNA codon shuffles down to the P site (well actually its the ribosome that moves in the 3’ direction) and so with each tRNA the ribosome moves one codon length along.

Question 39

When does the termination phase of translation occur? How does it work?

Answer 39

The termination phase of translation occurs when the ribosome moves along the mRNA in the 3’ direction until a stop/termination codon (UAA, UAG, or UGA) reaches the A site. The stop codon doesn’t bind to a tRNA and so doesn’t code for any amino acid, but triggers a release factor protein to bind
to the A site and disconnect the polypeptide from the tRNA in the P site. Now that P site is vacant, ribosome dissociates into start components.

Question 40

What are the key types of point mutation?

Answer 40

• Missense mutation (changes amino acid)
• Silent mutation (no change to amino acid)
• Nonsense mutation (inserts stop codon, shortens protein)
• Frameshift mutation (insertion/deletion of nucleotides, scrambles the following codons).

Question 41

What is a substitution or “point” mutation?

Answer 41

A substitution or “point” mutation changes a single nucleotide: changes one base. (Nucleotide=single base pair + sugar + phospate. 1 codon = 3 nucleotides).