Translation Flashcards

Question

There are differences between eukaryotic and prokaryotic mRNA. Tell me some things that eukaryotic mRNAs contain?

Answer 1

* a cap (made from a modified G nucleotide) and a poly(A) tail * No Shine-Dalgarno sequence * 5’ and 3’ UTRs may contain several features (sequence or structure-based) that help regulate protein expression/mRNA stability/localisation

Answer 2

* eIF3 equivalent to IF3, but 13 subunits * Binds eIF4G and the 40S subunit * PABP = poly(A)-binding protein

Answer 3

* Eukaryotic initiation differs considerably from bacterial initiation * **eIF2 +** the small 40S ribosome subunit + methionyl-tRNAimet + GTP bind to each other * The 40S subunit binds to mRNA with other initiation factors (eIFs) bound to the cap and **poly A tail** regions * The 40S ribosome then **“scans”** the mRNA looking for the AUG initiation codon usually uses first AUG it encounters * More efficient if this AUG is within the **Kozak Consensus** * eIFs then dissociate and the 60S subunit binds * Intact cap and tail regions are essential for initiation

Answer 4

* 48S pre-initiation complex (PIC) formation and mRNA circularisation * 40s ribosomal subunit * 48s is the whole unit

Answer 5

**Synergism between 5' and 3' ends** * mRNA transcribed in vitro, in combinations with or without m7G cap or poly(A) tail * Added to rabbit reticulocyte lysate (contains ribosomes and other factors required for translation) in presence of radioactive methionine * Products of in vitro translation separated by SDS-PAGE and visualized with autoradiography

Answer 6

* Many proteins can fold **spontaneously** using only the primary amino acid sequence information * Other proteins require assistance from **chaperones**

Answer 7

Chaperones – e.g., heat shock proteins – prevent illicit liaisons between proteins (e.g., interactions between exposed hydrophobic regions)

Answer 8

They are (hopefully) rapdily degraded

Answer 9

Protein misfolding in the ER can be a serious problem, and PERK exists to couple protein folding to protein synthesis

Answer 10

* Normally, **BiP** binds to PERK and keeps it in an inactive **monomeric** state * Can elicit **ER stress** using brefeldin A (blocks protein processing) or thapsigargin (interferes with ER Ca2+) * BiP **dissociates** to bind to unfolded proteins, activating PERK **dimers**

Answer 11

Suggests important role for PERK in cells that secrete a lot of protein e.g. pancreatic b-cells

Answer 12

Loss of PERK function – patients develop Type I diabetes, growth retardation, multiple other effects

Answer 13

* Translation is the synthesis of chains of amino acids into protein by decoding the message that was stored in the DNA (therefore change of “language” * mRNA is the intermediate, produced by transcription (nucleotide language remains similar) * Initiation is the positioning of the small ribosomal subunit at the initiation codon, and the joining of the large ribosomal subunit * Prokaryotic initiation uses rRNA:mRNA base pairing to define the start point, eukaryotic uses a sequence consensus * Eukaryotic initiation is more complex but more tightly controlled * Eukaryotic translation initiation is controlled by multiple mechanisms - Availability of cap-binding protein - Availability of ternary complex (contains first Met-tRNAi) - Proteins are sometimes also transported during translation - Accumulation of misfolded proteins causes activation of PERK * Next lecture – how amino acids are added to the correct tRNA

Answer 14

There are polar and non-polar amino acids. **Polar- hydrophilic** * Acidic: COO- provides the -ve charge * Basic: N+ provides the +ve charge * Uncharged polar: OH provides polarity **Non-polar- hydrophobic** * R group usually contains just C and H

Answer 15

This indicates that life arose from one original ancestor

Answer 16

A preference to decode certain codons and not others Important if making e.g. human protein in E. Coli

Answer 17

**triplet** **continuous** **non-overlapping**

Answer 18

If one RNA base specified one amino acid, only 4 amino acids could be encoded- one amino acid for each base

Answer 19

If a pair of any of the four RNA bases specified one amino acid, then only 16 amino acids (4²) could be encoded.

Answer 20

A triplet of any of the four bases can specify 64 combinations (43), therefore provides more than sufficient triplets to specify each of the 20 amino acids.

Answer 21

Addition or deletion of single nucleotide bases changes the frame downstream, therefore the code has to be **continuous**

Answer 22

The continued contact of tRNAs with the mRNA template during elongation steps means that the code cannot be overlapping

Answer 23

That more than one codon may specify a given amino acid

Answer 24

S, L and R

Answer 25

G, P, A, T and V

Answer 26

Isoleucine (I) and "stop"

Answer 27

F, D, N, E, Q, H, C, Y and K each pair either 'ends' with purines or pyrimidines

Answer 28

**silent mutations** in DNA/mRNA (usually the third position of a codon)

Answer 29

The arrangement of the code

Answer 30

70 and 90 bases

Answer 31

highly conserved tertiary structures (L-shape) with much internal base pairing

Answer 32

CCA Each amino acid is linked as an **ester** to the 3'-OH (or in some cases to the 2'-OH) of the last A

Answer 33

Numerous unusual post-transcriptionally **modified bases** tRNAs possess an **anticodon** that recognises the mRNA **codon** by anti-parallel base pairing

Answer 34

Its methylated so it doesn't look like a foreign invader to the body

Answer 35

**Pseudouridine (ψ)** has more possibilities for H-bonding (a,d) and more rigidity This is better for stability in tertiary structure and interaction with ribosome

Answer 36

The peptide bond -CO-NH- links amino acids and it is an **amide bond** and its synthesis requires the input of energy

Answer 37

Each of the 20 AA is first linked to the 3'-OH (or 2'-OH) of the terminal adenosine of a **specific tRNA** by an ester bond (gives an amino acyl-tRNA)

Answer 38

Peptide bond formation in the ribosome This ensures an amino acid is only linked to its correct tRNA, highly specific enzymes are used These vital enzymes are called **amino acyl-tRNA synthetases**

Answer 39

* tRNA synthetases recognise the tRNA and the amino acid * They catalyse the reaction of the terminal adenine ribose (of **CCA**) with the amino acid carboxyl group to give **NH₂CHRCO-^3'O-tRNA** * Two classes of synthetases exist (I and II)

Answer 40

1. Amino acid + ATP + Enzyme --\> Enzyme-AMP**-**Amino acid + PPi "activated intermediate" (**- = high energy bond)** 2. Enzyme-AMP-amino acid + tRNA --\> Aminoacyl-tRNA + AMP + Enzyme

Answer 41

The equivalent of **two ATPs** is used (the enzyme uses a second to drive the reaction)

Answer 42

1 in 10,000

Answer 43

**Proofreading-** mistakes made in reaction 1 are eliminated in reaction 2

Answer 44

in this example... * Phe cannot bind to the acylation site as it is to big * Alanine has managed to get into the acylation site as its small, however cannot bind to the hydrolytic site as the wrong tRNA has been made from when it bound to the acylation site so its not complementary * Val fits in both

Answer 45

The anticodon plays no role in loading of correct amino acid onto the tRNA but is essential for correct translation of code

Answer 46

then the amino acyl-tRNA still recognises the anticodon and inserts the wrong amino acid

Answer 47

Cysteine-tRNA^Cys is prepared

Answer 48

* **Raney nickel** (mix of Ni and Al) reduces cysteine’s -CH2-SH group **into -CH3 (alanine)** * Therefore cysteine-tRNA^Cys is converted into alanine-tRNA^Cys * The modified alanine-tRNA^Cys is used in the synthesis of haemoglobin in an in vitro experiment * The result is that alanine is incorporated into the haemoglobin in place of each cysteine (UGU or UGC codons) * Thus, the anticodon is recognised, not the amino acid (alanine) * Therefore accuracy/proofreading in aminoacylation of tRNAs is critically important

Answer 49

Its a term coined by Crick that defines the ability of the first base of an anticodon to base pair with **more than one** codon

Answer 50

An aminoacyl-tRNA to dock with more than one codon, so that fewer than 61 tRNAs are needed to decode the 61 codons

Answer 51

Wobble generally involved the nucleotides **U** or **inoside** **(I)** at the first position of the anticodon (**matches 3rd position of codon**)

Answer 52

**G** pairs with **C** **A** pairs with **U** **U** can also pair with **G**

Answer 53

**U** in the first anticodon position can base pair with **A** or **G** in the 3rd position of the codon

Answer 54

**G** in the first anticodon can base pair with **C** or **U**

Answer 55

**Inosine (I)** in the 1st position (5'- position)

Answer 56

guanosine without an amino group but is made by **deamination of adenine** This conversion is done AFTER transcription of the tRNA

Answer 57

A, U or C using 2 H bonds

Answer 58

If the first base of the anticodon is inosine (I) then it can base pair with A, U or zc Therefore, a single tRNA can recognise **More then one codon** e.g. the 5’-IGG-3’ anticodon of tRNAPro can recognise three of the proline codons

Answer 59

* To **activate** the amino acid as a tRNA ester * To act with the correct **synthetase** to ensure that the correct amino acid is linked to its tRNA * To base pair its **anticodon** triplet with the correct codon of mRNA - decoding role (anticodon:codon pairing can **“wobble”**) * To bring the correct amino acid to the **ribosome** * To facilitate **peptide bond** (-CO-NH-) formation of the nascent chain and the next amino acid

Answer 60

* Arrows show ribosomes on the Endoplasmic Reticulum making proteins that will be secreted or have roles in membranes * A single cell typically contains millions of ribosomes making 10 million peptide bonds per second per cell (in eukaryotes)

Answer 61

From the 5' to 3' end and proteins are synthesises starting from their **amino**-terminus to their **carboxyl** terminus

Answer 62

Occurs in ribosomes: complex **ribonucleoprotein** particles which bind mRNA and tRNA, and contain rRNA and proteins

Answer 63

over 2.5 million Da

Answer 64

In the **nucleolus**

Answer 65

* **30S** subunit – 16S rRNA + proteins S1- S19 * Contains the **“decoding centre” (DC)** * Helix 44 of 16S rRNA forms the **A** and **P tRNA binding sites** * **3’ of 16S rRNA** complements the Shine-Dalgarno in mRNA * **50S** subunit - 23S and 5S rRNAs + proteins L1– L31 * 23S rRNA forms 6 domains (I-VI) * Contains the peptidyl transferase centre (**PTC**) * Contains the peptide **exit tunnel**

Answer 66

Peptide bond formation

Answer 67

* Originally thought that rRNA was just a **scaffold** for the proteins * However, biochemical and genetic data pointed to a key role for **domain V** of 23S rRNA in the peptidyl-transferase function * Final confirmation that the ribosome is a **ribozyme (a ribosome capable of acting like an enzyme)** came from the crystal structure of the 50S subunit from the archaebacterium Haloarcula marismortui * **Ribozyme = an RNA molecule capable of acting as an enzyme**

Answer 68

* No ribosomal proteins located in the PTC (peptidyl-transferase centre) – nearest ones are **15-18Å** away – too far to take part * Ribosomes that are largely **depleted** of protein still have peptidyl-transferase activity

Answer 69

A (occupied by Aminoacyl-tRNA) P (occupied by Peptidyl-tRNA) E (occupied by deacylated tRNA, so Exit site)

Answer 70

Need the NH of incoming amino acid (attached to tRNA) to attack CO of the growing polypeptide, which is still attached to tRNA Ends up with extended polypeptide

Answer 71

* Need to break ester bond between amino acid and tRNA * Nucleophilic attack by amino group of the incoming amino acylated tRNA

Answer 72

Decreases the rate of peptide bond formation by roughly a million-fold

Answer 73

it does not

Answer 74

Ribosome helps to position substrates and water molecules with aids proton transfer and stabilisation of intermediates

Answer 75

it instead decreases activation energy needed for the peptide bond formation

Answer 76

* ensures **correct** amino acid is added sequentially to the growing protein chain by base pairing of transfer RNA (**tRNA)** with mRNA * decodes 10 – 40 aa per sec with only 1 in 10,000 error rates * already seen that accuracy of **charging** tRNAs with the correct amino acid is key to this, but elongation factors do check **fidelity** of codon: anticodon pairings

Answer 77

**A-site** aided by elongation factor **EFTu•GTP** If codon:anticodon is correct, EFTu·GTP is **hydrolysed** to EFTu·GDP EFTu·GDP is then released and **recycled** to EFTu·GTP by EFTs EFTs is a guanine nucleotide-**exchange** factor (GEF) EFTu·GDP + GTP --\> EFTu·GTP + GDP **Peptide bond** -CO-NH- formation occurs in the PTC

Answer 78

Translocation then occurs peptidyl-tRNA moves to the P-site; the spent tRNA moves to the E-site and **exits** the ribosome This requires EFG•GTP which is hydrolysed to EFG•GDP

Answer 79

The next aminoacyl-tRNA binds at A-site; cycle is repeated "costs" **2x GTP molecules**

Answer 80

Two steps 1. the 3'- ends of the tRNAs 2. Codon: Anticodon pairs

Answer 81

tRNA is **always** base pairs with mRNA, this prevents frameshifting, ensures **accuracy** and shows why code is read **triplet by triplet**

Answer 82

A "tunnel"

Answer 83

The ribosome exit tunnel This protects the new chain from inappropriate interactions and this allows it to sample multiple conformations

Answer 84

It **folding** and its final shape

Answer 85

**before** the C-terminal region is completed

Answer 86

Many proteins are initially synthesised as pre- or pro-proteins and are post-translationally modified(phosphorylation, glycosylation etc)

Answer 87

Coenzymes (NAD), Cofactors (Zn²⁺) or prosthetic groups (Haem)

Answer 88

**eEF1A** equivalent to EFTu **eEF1B** = EFTs (GEF for recycling eEF1A) **eEF2** = EFG

Answer 89

**Phosphorylation** of eEF2 (in response to rise in AMP, rise in Ca2+) reduces elongation rate

Answer 90

Until a **termination** codon occupies A-site

Answer 91

* Ribosome fails to find cognate AA-tRNA * Release factors RF1 or RF2 and RF3 bind (RF1/2 mimic tRNA) **- RF1, UAA/UAG** - **RF2, UAA/UGA** * The final aminoacyl-tRNA ester link is **hydrolysed** by reaction with H2O and the peptide is released from the ribosome * The mRNA and the ribosomal subunits **separate** from each other * RRF recycles the subunits

Answer 92

* eRF1 for all three stop codons, **eRF3** in the GTPase * several recycling factors **ABCE1** is key one

Answer 93

Because of small structural differences * **Chloramphenicol -** blocks peptidyl transferase * **Erythromycin -** blocks elongation by binding to the 23S rRNA tunnel * **Tetracycline -** prevents amino acyl tRNA binding

Answer 94

Aminoglycoside antibiotics paromomycin and streptomycin bind to decoding centre and induce errors in translation

Answer 95

A conformational change in 30s subunit, to closed state, activating hydrolysis of EFTu•GTP In the presence of paromomycin, even ‘near-cognate’ AA-tRNAs can induce this conformational change, so that mistakes occur more easily.

Answer 96

**Puromycin** - is an analogue of tyrosyl aminoacyl tRNA^tyr and causes premature peptide chain termination - also inhibits protein synthesis in eukaryotes Can use puromycin analogues as tags to assay protein synthesis

Answer 97

**_Summary_** * Ribosomes are complex molecular machines, with rRNA forming important structural parts e.g. a peptidyl transferase centre * PTC facilitates peptide bond formation between two charged tRNAs * Translation elongation and termination events in prokaryotes and eukaryotes are similar * Charging of tRNAs with correct amino acid ensures high fidelity * Fidelity of protein synthesis is also ensured by elongation factors which check mRNA:tRNA codon:anticodon interaction * Next lecture: what happens when silent or nonsense mutations occur in the open reading frame?

Answer 98

Remember that ribosome has three sites: * **A** (occupied by Aminoacyl-tRNA) * **P** (occupied by Peptidyl-tRNA) * **E** (occupied by deacylated tRNA, so Exit site)

Answer 99

* To create a peptide bond, need NH of incoming amino acid (attached to tRNA) to attack CO of the growing polypeptide, which is still attached to tRNA. * Ends up with extended polypeptide

Answer 100

* **eEF1A** equivalent to EFTu * **eEF1B** = EFTs (GEF for recycling eEF1A) * **eEF2** = EFG * **Phosphorylation of eEF2** (in response to rise in AMP, rise in Ca2+) reduces elongation rate

Answer 101

* Elongation continues until a **termination codon occupies A-site** * Ribosome fails to find cognate AA-tRNA * Release factors **eRF1 and eRF3 bind** (eRF1 mimics tRNA) * The final aminoacyl-tRNA ester link is hydrolysed by reaction with H₂O and the peptide is released from the ribosome * The mRNA and the ribosomal subunits separate from each other * ABCE1 recycles the subunits

Answer 102

* A surveillance mechanism to detect and destroy aberrant mRNAs containing Premature Termination Codons (PTCs) BEFORE translation * Prevents accumulation of proteins with C-terminal truncations, which could create inactive or even dominant negative versions

Answer 103

* Protein complex known as the **exon junction complex (EJC)** is deposited during splicing. * It is placed 20-24 nucleotides upstream of each splice site. * EJC contains various proteins, most important is UPF3 * During export UPF2 binds to UPF3 in EJC

Answer 104

* Duchenne muscular dystrophy (DMD) * Familial adenomatous polyposis * Hereditary breast and ovarian cancer * Polycystic kidney disease * Cystic Fibrosis PTCs may account for 90% of identified mutations in these diseases!

Answer 105

Gentamicin

Answer 106

PTC124 (Translaren/Ataluren) is available for DMD treatment in the UK, but CF trials failed, even though they showed promise in mice with G542X mutation of the CFTR

Answer 107

* Useful to stop ribosomes accelerating too quickly at start of ORF * Help to give enough time for the protein to fold * May be helpful in regulating protein expression * e.g. in ZEB2 translation of Leu(UUA)-Gly(GGU)-Val(GUA) causes ribosomal pausing and limits protein production but changing the codons to the common ones does not * BUT pausing can cause no go decay

Answer 108

* **Comparing the three surveillance mechanisms, all can lead to destruction of an aberrant mRNA** * **This is an important quality control mechanism** * **e.g., with No-Go decay, if the collided ribosomes do not dissociate, the reading frame can shift to the +1 frame and then make an aberrant protein**

Answer 109

This causes a reduction in protein synthesis of both host and viral mRNA, which should prevent further infection

Answer 110

poliovirus infection

Answer 111

Secondary and tertiary structures formed by base-pairing of single stranded RNA – depends on length and G-C content * Internal Ribosome Entry Sites (IRESs) * * May need non-canonical factors (ITAFs)

Answer 112

First discovered in picornaviruses, used to maintain synthesis of viral proteins at expense of host cell cap-dependent translation (Jackson 2005)

Answer 113

* Cellular mRNAs are degraded following infection, so there are less host cell messages to translate * This appears to be dependent on the viral non-structural protein 1 (Nsp1) * Even those transcripts which are produced in response to the infection are not translated * The Nsp1 blocks the mRNA channel of the 40S subunit * General protein synthesis is inhibited, including translation of genes of the innate immune response (e.g., IFN-β) * 10 of the 27 viral proteins bind to specific host RNAs (e.g., eEFs) * Nsp16 disrupts splicing * Nsp8 and Nsp9 stop N-terminal signal peptide of secreted/membrane proteins from being recognised

Answer 114

* mRNA surveillance acts as a final quality control step to ensure only a minimal amount of protein is translated from aberrant mRNAs * All mechanisms result in mRNA decay. * Nonsense mediated * Non-stop * No go * Viruses ensure their own protein synthesis by many mechanisms, which is one of the reasons it is so difficult to develop effective antiviral strategies * Many disrupt the host protein synthetic machinery directly, hijacking the translation to ensure viral proteins are made