protein synthesis

Question 1

protein synthesis components

Answer 1

The mRNA
tRNAs
Ribosome
Additional proteins

Question 2

genetic code rules

Answer 2

triplet code
3 bases code for 1 aa (codon)
It is non-overlapping
There is start and stop, but no other punctuation
It is degenerate
All codons have meaning (some mean same thing, synonymous)
1 start codon, 3 stop codons

Question 3

tRNAs

Answer 3

decode the sequence
tRNAs recognize codons in mRNA and carry the appropriate amino acid
Anti-codon in tRNA hybridizes with the codons in mRNA

Question 4

aminoacyl tRNA synthetases roles

Answer 4

attach the correct amino acid to its cognate tRNA
Cells have 20 different aminoacyl tRNA synthetases
Each synthetase must recognize the specific tRNA and the specific amino acid

Question 5

aminoacyl-tRNA steps (2)

Answer 5

First activate the amino acid

Transfer activated amino acid to tRNA

Question 6

2 classes of aminoacyl-tRNA synthetases

Answer 6

Class 1: glutaminyl-tRNA synthetase

Class 2: threonyl-tRNA synthetase

Question 7

Discrimination of various tRNAs by aminoacyl-tRNA synthetases

Answer 7

No universal rules
Some, but not all, use the anticodon
Bases in the acceptor stem can be important
Base at position 73 can be important (“discriminator base”)
The glutaminyl-tRNAGln synthetase makes extensive contacts with its cognate tRNAGln

Question 8

Codon : anticodon pairing

Answer 8

But polyU mRNA can interact with all Phe-tRNAPhe

So there must be more to codon-anticodon recognition

Question 9

wobble hypothesis

Answer 9

First 2 bases have stringent specificity, while third base pair is more flexible
Inosine, a base often found in the third position of the anti-codon, can form base pairs with cytosine, uracil, or adenine

Question 10

prokaryotic ribosomes composition

Answer 10

Ribosomes are proteins + RNAs

Ribosomes bring together the mRNA and amino acid-bound tRNAs and catalyze polypeptide synthesis

Question 11

30s rRNA precursor

Answer 11

includes rRNAs and tRNAs

RNaseIII and other nucleases release rRNAs and tRNAs

Question 12

tertiary structure of 16S rRNA

Answer 12

Folds into scaffold upon which proteins of 30S subunit can assemble
23S and 5S rRNAs establish the form of the 50S subunit
Each subunit can self-assemble after combining the individual proteins and rRNAs under appropriate conditions

Question 13

ribosomal subunit decoding center

Answer 13

is responsible for reading the mRNA by mediating codon-anticodon interactions
This is in small subunit

Question 14

ribosomal large subunit

Answer 14

Large subunit contains the peptidyl transferase activity needed to synthesize a new peptide bond

Question 15

both ribosomal subunits (large and small) are involved in…

Answer 15

translocation

Question 16

ribosome’s 3 binding sites for tRNAs…

Answer 16

A: acceptor site (binds incoming tRNA)
P: peptidyl site (binds growing polypeptide chain)
E: exit site (binds uncharged tRNA)

Question 17

translation overview

Answer 17

1) initiation
2) elongation
3) termination

Question 18

Initiation in bacteria

Answer 18

Proks use special initiator tRNA bound to formylated methionine
This tRNA can only be used for the first amino acid: can’t be used for internally coded methionine

Question 19

Formylation of methionyl-tRNAfMeti

Answer 19

The same aminoacyl-tRNA synthetase adds methionine to the Met-tRNAfMeti and the Met-tRNAMet
Formyl transferase recognizes the Met-tRNAfMeti
Formulation of Met blocks the 5’ amino group

Question 20

Identification of start codon in bacteria

Answer 20

5’ UTR of the transcript contains “Shine-Dalgarno” sequence, recognized by 3’ end of the 16S rRNA
Internal Shine-Dalgarno sequences may also be identified

Question 21

Initiation of protein translation (bacteria)

Answer 21

Requires initiation factors (IFs)

Question 22

elongation steps

Answer 22

1)Decoding: codon-directed binding of an incoming aminoacyl-tRNA in the A site
- Elongation factor EF-Tu-GTP facilitates recognition and binding of the incoming charged tRNA
- EF-Ts is a guanine exchange factor needed to recycle EF-Tu-GDP back to the EF-Tu-GTP form
2) Peptidyl-transfer: peptide bond formation in the A site
3) Translocation: The ribosome moves to the next codon in the mRNA
Elongation factor EF-G-GTP facilitates translocation

Question 23

decoding

Answer 23

Specific nucleotides in the 16S rRNA undergo conformational changes only when correct base-pairing occurs
If the appropriate conformational changes do not occur, the charged tRNA is ejected without hydrolysis of GTP

Question 24

peptidyl-transferase active site

Answer 24

Located in large subunit

Addition energy input is NOT needed for peptide bond formation

Question 25

peptide bond formation occurs where

Answer 25

on A site

Question 26

translocation sites

Answer 26

1) Deacetylated tRNA is moved from P-site to E-site 2) peptidyl-tRNA in A-site must be moved to P-site This opens A-site for next charged tRNA 3) Translocation is a 2-step process The acceptor stems in the 50S subunit move before the anticodon stems in the 30S subunit move Hydrolysis of EF-G-GTP is needed for second step

Question 27

termination

Answer 27

Elongation continues until a stop codon is reached RF-1 binds UAA and UAG RF-2 binds UAA and UGA RF-3 is recruited Binding triggers hydrolysis of nascent peptide RRF triggers release of mRNA from the ribosome

Question 28

role of GTP hydrolysis in protein synthesis

Answer 28

Energy of GTP hydrolysis fuels conformational changes in ribosome that drives the steps of protein synthesis During elongation, 2 GTP are hydrolyzed for each amino acid incorporated into the growing polypeptide chain 1 GTP is also needed during initiation, plus 2 GTPs needed for termination

Question 29

release factors

Answer 29

mimic structure of elongation factors | IF-2, EF-Tu, EF-G, and RF-3 all bind the same place on the ribosome

Question 30

protein synthesis in euks

Answer 30

Euk mRNA: 5’ cap, 5’ UTR, coding sequence, 3’ UTR, polyA tail 5’ cap is needed for recognition by the ribosome (no Shine-Dalgarno sequence) Only one coding sequence can be read per mRNA (monocistronic)

Question 31

euk translation initiation (3 stages)

Answer 31

1) 43S preinitiation complex 2) 48S initiation complex 3) 80S initiation complex

Question 32

mRNAs do what prior to translation

Answer 32

circularize

Question 33

peptide chain elongation and translation in euks

Answer 33

Similar to elongation in proks, just different names eEF1 is made of eEF1A (~EF-Tu) and eEF1B (~EF-Ts) eEF2 (~EF-G) Euk cells have a single release factor (RF)

Question 34

When is controlling protein synthesis important?

Answer 34

If there is a backlog of normal protein folding, protein translation slows Many antibiotics preferentially inhibit protein translation in bacteria

Question 35

Controlling translation initiation

Answer 35

Phosphorylation of eIF2 prevents eIF2B from exchanging GDP for GTP This slows translation by inhibiting initiation There are several eIF2alpha kinases that become active under specific cellular conditions