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Biochem > Protein Synthsis Steps > Flashcards

Protein Synthsis Steps Flashcards

1
Q

Charged tRNAs

A

Aminoacylated

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2
Q

Enzyme for connecting tRNAs to matching amino acids

A

Aminoacyl-tRNA synthetase

Each enzyme is specific for only one amino acid

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3
Q

2 classes of synthetases

A

Different pathways for connecting amino acids

Each group helps about half of the amino acids

No clear common ancestor

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4
Q

Generic reaction catalyzed by aminoacyl synthesis

A

Amino acid + tRNA + ATP (Mg2+) —> aminoacyl-tRNA + AMP + PPi

Steps

Amino acid interacts with atp with carboxyl group oxygen as a nucleophile and and alpha phosphate of atp acceptor —> releases PPi

Aminoacyl-AMP enzyme bound intermediate is formed

Aminoacyl group transferred from intermediate to tRNA
Ester linkage betweeen tRNA and AA

Class I
Adenine terminal of tRNA has 2’ o attack carboxyl c —> releases amp —> flipped to 3’o

Class II
2’o attacks initially and relases amp

Product = aminoacyl-tRNA

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5
Q

Synthetase proof reading

A

Many but not all synthetases have proofreading abilities

Will have two active sites

One for both correct and incorrect to attach to

1 that only incorrect will attach to and be hydrolyzed

Additionally, synthetases hydrolyze ester linkages and this hydrolysis is accelerated when tRNA incorrectly charged

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6
Q

Error rate for protein synthesis

A

1 mistake per 10^4 correct

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7
Q

What is the second genetic code?

A

Synthetases’ need to identity amino acids and tRNAs

ID primarily by info in AA arm and anticodon arm

Class I and class II bind to opposite faces of substrate tRNA

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8
Q

For genetic code expansion:

A

Need: - new syntheses - new AA - new cognate tRNA

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9
Q

2 extra amino acids

A
  • Selenoysine
    = normal ser attached to tRNA but edited before ribosome
  • pyrrolysine
    =unique tRNA and unique synthetase
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10
Q

Unnatural genetic code expansion

A

-geneses for tRNA and synthetase taken from certain organism
tRNA selected by anticodons don’t determine having synthetase help reaction so codon can be changed

Codon changed to least common stop anticodon = CUA

Need to make sure endogenous synthetase doesn’t work on tRNA so put in plasmid along with another plasmid that has Barnard gene
If tRNA is used to translate Barnard gene functionally rather than as a series of stop codons, the. Endogenous synthetase is working = dead cell bc Barnard deadly

= alive good for more testing = negative selection

Need to make sure that modified synthetases are specific only modified tRNAs

1 plasmid has both synthetase genes and B lactase gene
Other has tRNA gene edited with lots of UAG

If lac expressed then can live on ampicillin

Which is proof of tRNA and synthetase interaction

If it could interact with old tRN for UAG, b lac could not be expressed

= positive selection

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11
Q

All organisms have one codon but two tRNAs for what codon?

A

AUG (Met)

One for initiation
One for met within polypeptide

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12
Q

Bacteria name different for two different aug codons

A

fMet and Met

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13
Q

Bacterial initiation mechanism

A

2 steps

Methionine + tRNA + ATP —> met-tRNA^fMet + AMP + PPi

N-formeletetrahydrofolate + met-tRNA^fMet —> fmet-tRNA^fMet + tetrahydrofolate

Via transformylase

This can now no longer be added to internal peptide due to N formyl physical blockage

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14
Q

Eukaryotic cells initiate with

A

Met reside but two different tRNAs depending on initiation or peptide bonding

BUT mitochondrion and chloroplasts do use fMet

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15
Q

IF1

A

Prevents premature binding to A site

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16
Q

IF2

A

Facilitates fMet-tRNA^fMet binding to 30S subunit

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17
Q

IF3

A

Prevents premature 30s and 50s subunit interaction

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18
Q

ElF1A (eukaryotic)

A

Prevents premature A site binding

IF1 homolog

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19
Q

ElF1 (eukaryotic)

A

Binds to 40S E site
Helps tRNA-elF2-GTP complex bind to 40S

20
Q

elF2 (eukaryotic)

A

GTPase

Helps 40S + met-tRNA^Met binding

21
Q

elF2B (eukaryotic) + elF3 (eukaryotic)

A

1st to bind to 40S subunit
Help with all subsequent steps

22
Q

elF4E (eukaryotic)

A

5’ cap of mRNA binding
Mediates association with 43 S complex

23
Q

elF4A (eukaryotic)

A

ATPase
RNA helicase
Removes secondary structure of mRNA for 40S binding

24
Q

elF4G (eukaryotic)

A

Binds to polyA binding protein to help with mRNA circularization

Linker protein

Works either elF4E and elF3 to form first link between 40s subunit and mRNA

25
Q

elF4F (eukaryotic)

A

Complex with elF4E elF4G, elF4A

26
Q

elF4B (eukaryotic)

A

Binds to mRNA
Helps scan for AUG

27
Q

elF5 (eukaryotic)

A

Promotes initiation factors dissociation from 40S —> prepping of 80S

28
Q

elF5b (eukaryotic)

A

GTPase that promotes dissociation of initiation factors prior to final ribosome assembly

Homolog to IF2

29
Q

Bacterial initiation

A

30S + if1 + if3

30s complex + mRNA
=alignment via Shane dalgarno sequence
=8-13 bps A + G of 5’ mRNA end
Align with c + u in 16S rRNA

Initiating AUG = at P site to which fMet - tRNA ^fMet binds

This complex bindss with 50S subunit

IF2 hydrolzyed and uses up GTP

Initiation factors released

=70S initiation complex

Matching = codon anticodon interaction, tRNA fitting into p site, Shane dalgarno for mRNA alignment

30
Q

3 types of elongation factors

A

EF-Tu
EF-T
EF-G

31
Q

EF-Tu

A

EF-Tu-GTP complex helps aminoacylated tRNAs bind to A site of 70S initiation complex

Binding releases EF-Tu- GDP and PPi

EF-T comes to bind to EF-Tu to release GDP

recycled when EF-Tu replaces EF-T with GTP

32
Q

Initial peptide bond formation

A

Transfer of N-formylmethionine group connected to fMet tRNA in p site to amino group of amino acid at A site

N of AA is nucleophile to carboxylic Carbon

Produces h2o

33
Q

Initial peptide bond formation

A

Transfer of N-formylmethionine group connected to fMet tRNA in p site to amino group of amino acid at A site

N of AA is nucleophile to carboxylic Carbon

Produces h2o

34
Q

EFP

A

Binds E and Pnsites for stability esp during double proline addition bc side chains are tricky

If efp lacking - lots of ribosomal stalling = health issues

Eukaryotic homolog: elF5A

35
Q

TmRNA and SmpB

A

Transfer messenger RNA and small protein B

Part of trans-translocation system that saves translation when mRNA stops before stop codon

TmRNA binds to A site until stop codon within tmRNA found and ribosome is recycled

36
Q

Peptidyl transferase

A

Catalyzes peptide bond formation
Within 23S rRNA
Aligns with tRNA and a and p sites

37
Q

EF-G

A

AKA translocase

Helps shift over mRNa
Needs GTP energy

38
Q

Allosteric change during translocation

A

New AA in A site leads to structure change that forces uncharged tRNA from E site

39
Q

Proofreading and EF-Tu

A

Time for association and release of GDP = time to check codon and anticodon fidelity

(Cannot double check correct AA is on tRNA)

40
Q

Termination codons

A

UAA
UAG
UGA

41
Q

RF1

A

Recognizes UAG and UAA

42
Q

RF2

A

Recognizes UGA and UAA

43
Q

RF1 and RF2

A

Have polypeptide transfer to h2o molecule

44
Q

RF3

A

Thought to belong ribosomal subunit release

45
Q

Ribosomal recycling

A

EF-G + RRF
GTP is hydrolyzed and they are released

Replaced by IF3
= tRNA dissociation

46
Q

Polysome

A

Large cluster of ribosomes

In bacterial
Same mRNA that is still being transcribed

Biochem (23 decks)

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