L5 From RNA to Protein

Question 1

two regions of tRNA important for translation

Answer 1

first region forms anticodon to match with mRNA

second region is a short single strand at 3’ end of trna molecule > place where amino acid that matches codon is attached to tRNA

Question 2

what is a wobble position

Answer 2

first 2 bases of a codon must pair exactly with anticodon > 3rd base (wobble position) does not need to match directly > helps trna recognise multiple codons coding for the same amino acids > reduce no. of trna needed

Question 3

possible anticodon bases for wobble codon base U and C in eukaryotes

Answer 3

U: A, G or I

C: G or I

Question 4

why do trna have inosine as a base

Answer 4

often found only at wobble position

allows for more flexible base pairing with codon > enable single trna to recognise multiple codons

Question 5

process of amino acid activation by synthetase enzymes

Answer 5

1. COOH group of a.a attached to AMP > form adenylate amino acid (process driven by hydrolysis of ATP)
2. without leaving synthetase enzyme, AMP-linked COOH on a.a transferred to -OH group on sugar at 3’ end of trna molecule via activated ester linkage > form aminoacyl-trna molecule

different amino acids have different synthetases

Question 6

how does synthetase enzyme select the correct a.a via two-step mechanism

Answer 6

1. correct a.a has highest affinity for active-site pocket os its synthetase > favoured over other a.a > other a.a larger than correct a.a excluded from active site
2. after trna binds > enzyme tries to force adenylated a.a into editing pocket > correct a.a cannot fit > only wrong a.a fit inside > removed from AMP by hydrolysis to remove the incorrect a.a

Question 7

how are amino acids connected to form polypeptide during translation

Answer 7

formation of peptide bond between carboxyl group (C-terminal) at end of growing chain and free amino group (N-terminal) of incoming a.a

throughout process > growing COOH end remains activated by covalent attachment to trna > each addition of a.a disrupts high energy covalent linkage > immediately replaces it with identical linkage on most recently added a.a

Question 8

subunits of eukaryotic and prokaryotic ribosomes

Answer 8

eukaryotic (80S): 60s (large subunit) and 40s (small subunit)

prokaryotic (70s): 50s (large subunit) and 30s (small subunit)

Question 9

function of small and large subunits of ribosomes

Answer 9

small: ensures each mrna is read correctly and paired with the appropriate trna

large: catalyses formation of peptide bonds between a.a

when not actively synthesising proteins, two subunits are separate > only join together on mrna (5’ end) during initiation of translationr

Question 10

rate of translation of eukaryotic and prokaryotic ribosomes

Answer 10

eukaryotic: adds 4 a.a to chain per second

prokaryotic: adds 20 a.a per second

Question 11

how many binding sites are there in a ribosome

Answer 11

4, one for mrna, the other 3 (A, P and E site) for trna

A: aminoacyl site
P: peptide site
E: exit site

Question 12

process of adding amino acids to growing polypeptide chain

Answer 12

1. trna carrying next a.a binds to A site by forming base pairs with mrna codon
2. COOH end of chain released from trna at P site and joined to free amino group of a.a linked to trna at A site > form new peptide
3. large subunit moves relative to mrna held by small subunit > shifts trna from P to E site and A to P site
4. another series of conformational changes moves small subunit and its bound mrna 3 nucleotides > ejecting trna from E site > reset ribosome to receive next aminacyl-trna

Question 13

what is one additional feature that makes translation especially efficient and accurate

Answer 13

two elongation factors enter and leave ribosome during each cycle > each hydrolysing GTP to GDP and undergoing conformational changes

bacteria: EF-Tu and EF-G

eukaryotes: EF1 and EF2

Question 14

how does EF-Tu help ribosome in elongation of chain

Answer 14

EF-Tu binds to aminoacyl-trna > carry to ribosome

if anticodon of trna matches mrna > EF-Tu hydrolyses GTP to GDP and detaches > allow trna to stay

if trna does not match > leaves and another trna tries again

Question 15

2nd opportunity for ribosome to prevent incorrect a.a from being added to growing chain

Answer 15

after EF-Tu leaves, ribosome has a brief pause > if trna is incorrect > more likely to fall off before new a.a is added

based on the fact that time delay is shorter for correct than incorrect codon-anticodon pairs, and mismatches trnas dissociate more rapidly than those correctly bound

Question 16

difference between EF-Tu and EF-G

Answer 16

EF-Tu: delivers aminoacyl-trna to ribosomal A-site > induce long and short range conformational change > dissociate after GTP hydrolysis

EF-G: binds to ribosome after EF-TU dissociates and use energy to push it 3 nucleotides forward

Question 17

how does ribosome act like an enzyme

Answer 17

made up of 2/3 rna and 1/3 protein > rrna is plays the main role of catalysing protein synthesis, not the protein

Question 18

how does ribosome catalyse reactions without metal ions

Answer 18

normally metal ions help rna carry out chemical reactions

in ribosome, 23s rrna creates special pocket that carefully positions reactants > precise positioning speeds up reaction

Question 19

how does trna help in catalysis

Answer 19

trna in P site has OH group that participates directly in catalysis > ensures reaction only happens when trna is correctly placed in ribosome

Question 20

function of ribosomal proteins in ribosomes

Answer 20

to stabilise rna core while permitting changes in rna conformation

Question 21

how is translation initiated

Answer 21

initiator trna always carry anticodon complementary to start codon AUG (methionine for eukaryotes, formylmethionine for prokaryotes) > loaded into small subunit along with initiation and elongation factors

small submit binds to 5’ end mrna (recognised by 5’ cap) > moves along mrna to search for first AUG (additional initiation factors that act as ATP powered helices facilitate the movement)

imitation factors dissociate > large subunit assemble with complex > initiator trna remains at P site > leave A site empty for protein synthesis to begin

Question 22

what is Kozak sequence

Answer 22

5’ ACCAUGG 3’

sequence of nucleotides around AUG affects how ribosome recognises it

perfect Kozak sequence or similar to Kozak > translation starts > if different then ribosome skips first AUG and find the next AUG

Question 23

how is mechanism for selecting start codon in bacteria different from eukaryotes

Answer 23

bacteria dont have 5’ cap to signal ribosome where to start

each bacterial mrna have ShineDalgarno sequence located few nucleotides upstream of AUG > forms base pair with 16s rrna of small subunit to position AUG in ribosome

Question 24

how is the end of translation marked

Answer 24

by stop codons UAA, UAG, UGA

not recognised by trna and do not specify a.a > simply to signal ribosome to stop translation

release factors bind to ribosome with stop codon positioned at A site > peptide transferase catalyse addition of water molecule instead of a.a to peptide-trna > frees COOH of growing chain > polypeptide chain released

Question 25

how does growing polypeptide chain move through the ribosome

Answer 25

moves through large, water filled exit tunnel in large subunit walls of tunnel primarily made of 23s rrna > patchwork of tiny hydrophobic surfaces embedded in more extensive hydrophilic surface > ensure surfaces do not interact strongly with growing chain

Question 26

why are proteins made on polyribosomes

Answer 26

multiple invitations take place on each mrna for multiple ribosomes to help translate mrna at the same time as soon as preceding ribosome translated enough > 5' end of mrna threaded into new ribosome bc of this > most mrna found in form of polyribosomes (large cytoplasmic assemblies made up of several ribosomes spaced as close as 80 nt apart along single mrna molecule