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BIOC2001 TEST TWO > Translation - Professor Gout > Flashcards

Translation - Professor Gout Flashcards

1
Q

How many proteins are in the Human proteome and why is this so much larger then the amount of genes in the genome?

A

There are around 20,000 genes in the human genome with more than 300,000 proteins in the proteome. This is due to RNA splicing where a single gene can make many slightly different mRNAs and therefore proteins.

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2
Q

What three things do post-translational modifications do to a protein?

A
  • Sub-cellular location
  • Activity
  • Stability
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3
Q

Does DNA, mRNA and proteins all have half-lives?

A

DNA does not
Both mRNA and proteins do, once mRNA is transcribed it does not stay around for long and proteins are constantly being synthesised and degraded.

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4
Q

Give a few examples of things that can change protein biosynthesis

A

Nutrients, Hormones, Growth factors, Pathogens, Stress

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5
Q

Give a few examples of what change of protein biosynthesis can do to a cell

A

Proliferation, Apoptosis, Growth, Differentiation, Transformation

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6
Q

Explain the phrase “Gene expression is cell specific”

A

All the cells in your body have the same DNA but they look and function differently depending on their cell type as they are transcribing different mRNA. They have different phenotypes that are mediated through different sets of proteins, they express different proteins to do their specific tasks.

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7
Q

What are the 3 stop codons?

A

UAA, UGA, UAG

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8
Q

What is so significant but the codon AUG?

A

This codon is the start codon and it codes for the amino acid methionine

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9
Q

What does a degenerate genetic code mean?

A

A single amino acid can be specified by more then one codon, with the exception of Methionine and Tryptophan

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10
Q

What is a Ribonucleoprotein?

A

Ribonucleoproteins are proteins that have a specific structure to interact with RNA and protect them from degradation, they cover the RNA. mRNA is protected in the cytoplasm by being a ribonucleoprotein particle. Ribonucleoproteins also contain information of where the RNA should go such as to the ribosome or the synaptic cleft.

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11
Q

What does a 5’ cap do?

A

It protects the mRNA from degradation by nucleases in the ctyoplasm

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12
Q

What does the 3’ Poly A tail do?

A

It protects the mRNA from exonucleases in the cell

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13
Q

What can mRNA do when associated with the ribonucleoprotein complex?

A

mRNA can attach to cellular structure, like actin filaments and synaptic structure and therefore mRNA can be specifically localised in a cell.

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14
Q

Do mRNA have internal sequences for their own degradation?

A

Yes, however they are usually stable in the cell for several hours

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15
Q

What does UTR stand for?

A

Untranslated region

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16
Q

How many tRNAs are there in eukaryotes?

A

About 100

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17
Q

What is at the 3’ end of a tRNA?

A

CCA sequence to which amino acids are linked

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18
Q

What three structures on a tRNA molecule are important in regulating amino acid loading?

A

Anticodon - Hybridises with the codon on the mRNA
Acceptor Stem - Where acceptation takes place
Discriminator Stem - Discriminates which amino acid is bound to the tRNA

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19
Q

What is the discriminator base?

A

The discriminator base is crucial for specific recognition of the correct amino acid

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20
Q

What reaction does the enzyme tRNA synthetase catalyse?

A

The reaction of an adenylated amino acid (phosphate added on using an ATP also done by tRNA synthetase) and a tRNA to produce an aminoacyl-tRNA. A high energy ester bond holds the amino acid to the tRNA.

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21
Q

What happens to an amino acid when in complex with a tRNA?

A

The amino acid is activated and can participate in peptide bond formation

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22
Q

What is the Wobble hypothesis?

A

There are 61 possible codons but only 20 tRNAs. The wobble hypothesis states that the same aminoacyl-tRNA recognises multiple codons, so fewer tRNAs are required to translate the genetic code. The 3rd base in the triplet codon can hydrogen bond with different bases that don not need to be its Watson-Crick pair.

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23
Q

What is the most abundant RNA-protein complex in the cell?

A

The ribosome, ribosomal proteins are important for maintaining the stability and integrity of the ribosome, but not for catalysis. rRNA acts as a ribozyme and has the catalytic activity.

24
Q

What are the two subunits of eukaryotic ribosomes?

A

40S and 60S and they can be separated using ultracentrifugation

25
Q

Are ribosomes constantly present in cells?

A

No, if protein translation is not needed then ribosome production will decrease and vice versa

26
Q

What do E,P and A sites stand for?

A

Exit site
Peptidyl transferase site
Amino acid site

27
Q

Where does Ribosome assembly take place?

A

In the nucleolus

28
Q

In prokaryotes, what is a Shine-Dalgarno sequence?

A

It is a sequence that only occurs in prokaryotes and it defines the start codon. So if a prokaryotic mRNA had lots of AUG codons in it the ribosome will not know which one is the start codon. The shine-Dalgarno sequence marks the correct AUG as it is 5-9 bases before the correct AUG start codon.

29
Q

How is the start AUG codon recognised in Eukaryotes?

A

By a sequence called a Kozak sequence, this sequence is slightly different from the Shine-Dalgarno because the sequence is not outside of the AUG codon it is around it.

30
Q

What is the difference between monocistronic and polycistronic mRNA?

A

Monocistronic mRNA is in eukaryotes, this means that an mRNA has one start and one stop codon with one ORF.

Polycistronic mRNA is in prokaryotes, the same mRNA can code for many different proteins with multiple start and stop codons and different ORFs. This is beneficial to prokaryotes as transcription only needs to occur once to make lots of proteins. Eukaryotes do not use this system as they need to regulate translation to a much higher degree then prokaryotes.

31
Q

What a polysome?

A

When many ribosomes come together and translate of one mRNA, to create many different proteins

32
Q

What are the 6 most common post-translational modifications? And describe them

A
  • Phosphorylation, the most abundant post-translational modification and it occurs in the SER on Ser, Thr and Tyr.
  • Methylation - occurs on Arg and Lys and controls the process of gene expression at the level of transcription
  • Acetylation - occurs on lys, the opposite of methylation, when this group is added (especially to histones) it unwinds DNA so RNA polymerase can access it
  • Ubiquitination - occurs on Lys, and targets a protein for degradation
  • Glycosylation - it is a signalling molecule that is usually added to membrane proteins. O-linked version occurs on Ser and Thr and N-linked version occurs on Asn
  • Hydroxylation - occurs on Pro and Lys not that important, just an addition of OH
33
Q

How does a protein know where to travel to where it is needed?

A

After translation, proteins have a specific sequences of amino acids that indicate where the protein needs to go.

34
Q

What are the differences in eukaryotic and prokaryotic translation?

A

In prokaryotes there is coupled, transcription and translation as there is no organelles and compartmentalisation. Also there are no Introns in prokaryotes with little regulation of the process.
In eukaryotes there is much more regulation, transcription and translation are not coupled as transcription occurs in the nucleus and translation occurs in the cytoplasm and they are separated by a membrane. mRNA needs to go through lots of post transcriptional steps to allow it to go into the cytoplasm to be translated. 5’ cap, 3’ Poly A tail and splicing. The mRNA is then bound to many proteins to protect it and direct it to where it needs to go.

35
Q

What are the three main steps in protein synthesis?

A

Initiation
Elongation
Termination

36
Q

What is the rate limiting step of translation?

A

Initiation, it requires the hydrolysis of both ATP and GTP. It is the most highly controlled step

37
Q

What are the two mechanisms of initiation of translation?

A

Cap-dependent initiation

Internal ribosome entry site (IRES)

38
Q

Describe cap-dependent initiation

A

The initiation complex interacts with the 5’ cap structure and scans in the 5’-3’ direction until the start AUG codon is found, in this mechanism the 5’ cap is needed for the mRNA to be translated

39
Q

Describe Internal ribosome entry sites (IRES)

A

The initiation complex binds upstream of the AUG codon. This mechanism occurs when there is no 5’ cap on the mRNA, the ribosome does not start at the 5’ cap it starts in the 5’ UTR.

40
Q

Describe Cap- dependent initiation in detail

A

Initiation factor eIF4E recognises the 5’ cap on the mRNA. eIF4E then binds to the 5’ cap, once it has bound it recruits another initiation factor eIF4G. eIF4A and eIF4B then bind to the large scaffold protein eIF4G. PABP then interacts with eIF4G and PABP also interacts with the polyA tail on the 3’ end of the mRNA. This circularises the mRNA making it easier to be translated. This mechanism ensures only transcription of intact mRNA with both 5’ cap and 3’ polyA tail.
The first amino acid is methionine, the initiation factor eIF2 recognises and binds to the tRNA with methionine. eIF3 recognises eIF2 and binds to it and also binds to eIF4G. eIF5 is bound to the 40S ribosomal subunit and eIF5 also binds to eIF3. This creates a huge complex on the mRNA. Then scanning occurs.

41
Q

What happens once the Kozak sequence containing the AUG codon has been found?

A

The Methionine tRNA will recognise the AUG start codon, 60S ribosomal subunit now binds. There is a final checkpoint here where eIF5 checks that the correct AUG codon is in place before the 60S ribosomal subunit binds. This is the end of initiation.

42
Q

Explain the elongation cycle in eukaryotic protein synthesis

A

EF-2-GTP enters the ribosome, it is hydrolysed into EF-2-GDP and the energy from this hydrolysis pushes the new tRNA into the P site and the deacylated first tRNA in the E site. EF-2-GTP translocates the polypeptide chain from the P site to the A site, the tRNA now moves into the P site and the A site is free.
EF-1A-GTP is an exchange factor that brings new tRNAs into the A site.
The cycle is repeated.

43
Q

Briefly explain the termination of transcription

A

Releasing factor eRF1 structurally mimics tRNA that is bound to EF-1A-GTP. eRF1 fits into the ribosomal A site where is recognises the stop codon. It then releases the completed polypeptide by catalysing a nucleophilic attack on the ester bond between the peptide and the P-site.

44
Q

How does eRF1 mimic tRNA?

A

The shape of eRF1 is the same as tRNA, only the releasing factor will recognise the stop codon as there is no tRNA that will recognise a stop codon. The catalytic activity of eRF1 is stimulated by the GTP-bound form of another releasing factor eRF3. eRF1 has three domains. Domain 2 recognises the stop codon. Domain 3 catalyses the hydrolysis of the completed peptide form the P-site. Domain 1 is the anticodon like structure.

45
Q

What are the three release factors in prokaryotes and what do they do?

A

RF-1 - operates with UAA and UAG stop codons
RF-2 - operates with UAA and UGA stop codons
RF-3 - is a ribosomal-dependent GTPase that helps RF-1 and RF-2 bind to the ribosome

46
Q

What are the two release factors in eukaryotes and what do they do?

A

eRF1 - operates with all three stop codons

eRF3 - performs the role of bacterial RF3

47
Q

Name the six ways in which protein translation is controlled

A
  • mRNA level/ stability
  • Amino acid concentration
  • Ribosomal Biogenesis
  • Initiation factors
  • Elongation factors
  • Termination factors
48
Q

Why do RNAs travel around the body?

A

It is easier to transport small mRNAs around the body compared to huge proteins, this is why RNA is moved to different locations in the body and then translated once there.

49
Q

What is an exon junction and why is it important?

A

The location where two Exons are joined together during post-translational modifications. There complexes in the mRNA tell the ribosome if it has finished translating yet or not.

50
Q

How are mRNAs with premature stop codons targeted for degradation?

A

They are targeted for nonsense mediated degradation. If a premature AUG codon is reached the ribosome will be aware that it is premature due to the exon junction complexes. The cell knows that there is a junction that it has not reached yet as it has sensed upstream factors, these instruct the ribosome to tell it that this stop codon is premature. The ribosome dissociates and exonucleases will binds to the 5’ end and then degrade the mRNA. Before this degradation can occur though the 5’ cap needs to be removed.
Upf (upstream factor proteins) bind to the ribosome to tell it that the codon is premature and they also activate the decapping enzyme. Which removes the 5’ cap so degradation can occur.

51
Q

How are mRNAs with no stop codons targeted for decay?

A

The ribosome will eventually start translating the polyA tail and this codes for lysine. A protein called Ski7 specifically recognises this sequence and binds, this allows other proteins to interact to form the exosome complex. 3’ exonucleases and a lysine specific protease enzyme are added to the structure. The faulty mRNA and the faulty protein are chopped away and the materials are used again.

52
Q

What is a frameshift mutation?

A

When a base is missed out and this moves the ORF down by one base and so all the codons will be different and different amino acids will be incorporated into the protein

53
Q

What is a missense mutation?

A

When a base is substituted for another base, can code for a new amino acid or a premature stop codon.

54
Q

How does the body inhibit translation during a viral infection?

A

Most Viral mRNAs have a 5’ cap. Viral infections cause the body to produce interferons as part of the innate immune response. Interferons bind to cell surface receptors and activate transcription of antiviral genes. Two interferon induced genes are key players in down regulation of translation.
RNase L - degrades RNA
PRK (Protein kinase RNA-activated), PRK phosphorylates eIF2A and this inhibits the initiation of translation. When eIF2A is phosphorylated it reacts better however it cannot dissociate and this inhibits translation.

55
Q

How does the body inhibit translation during piconorovirus infection?

A

5’ is not present in the mRNA of piconorovirus so it wants to be translated using IRES. In an infected cell the body cleaves the C terminus off the eIF4G initiation factor, this is crucial in recruiting the 40S ribosomal subunit.

56
Q

Why are hepatitis C mRNAs special?

A

Only one initiation factor is needed to start translation, eIF3. eIF3 mediates the interaction between the viral IRES mRNA and the 40S subunit. A dramatic change in the conformation of the 40S subunit occurs when it binds to Hepatitis C virus IRES setting the AUG at the p site.

57
Q

Name four antibiotics and how they work to inhibit bacterial translation

A

Puromycin - Aminoacyl-tRNA analogue, causes premature chain termination
Tetracycline - Inhibits aminoacyl-tRNA binding to the A-site
Chloramphenicol - Inhibits peptidyl transferase activity
Erythromycin - Binds peptidyl transferase and blocks polypeptide translocation.

BIOC2001 TEST TWO (9 decks)

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