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Biochemistry I > Genetic code + translation > Flashcards

Genetic code + translation Flashcards

1
Q

What are pri-miRNAs?

What is their function?

A

primary transcripts miRNAs transcribed by RNA pol II

  • promoting mRNA degradation
  • stimulating poly-A-tail degradation
  • inhibition of translation
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2
Q

What is a codon?

How many are there?

A

three letter base code w/ 64 possibilities

→ coding for 20 different amino acids

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3
Q

List the 5 features of the genetic code.

A
  • degenerate
  • unambiguous
  • non-overlapping
  • non punctuated
  • universal

DUNo NpU

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4
Q

The genetic code is non-punctuated.

What does it mean?

A

translated continuously until stop codon reached, no bases skipped

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5
Q

The genetic code is non-overlapping.

What does it mean?

A

each nucleotide is part of only 1 codon and only read once

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6
Q

The genetic code is degenerate.

What does it mean?

A

18 of 20 AAs coded by more than 1 codon

only exceptions: Met and Trp

(most are 4-fold degenerate at 3rd position)

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7
Q

The genetic code is almost universal.

What does it mean?

A

most codons code for the same AA in different organism

BUT: not true for mitochondria

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8
Q

The genetic code is unambiguous.

What does it mean?

A

each triplet nucleotide codon in mRNA codes only for 1 specific AA

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9
Q

Which codons code start and stop signals for translation?

A
  • AUG → start signal, codes for Met
  • UAA, UAG, UGA → stop signal to terminate polypeptide translation
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10
Q

There are 2 amino acids which are not directly coded for in the genetic code?

Which amino acids and how are they translated?

A

2 stop codons:

  • UGA → selenocystein
  • UAG → pyrrolysine (not present in humans)

efficiency of translation of those 2 AAs depends on protein that is synthsized in translation initiation factors

just know, wiki says UAG codes for pyrrolyisine, lecture says UGA does

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11
Q

What is the function of tRNA?

A

each tRNA has specific anticodon and carries an AA that is specific for its anticodon

→ use ribosome to bind to complementary codons on mRNA, then transfer their AA to form polypeptide

⇒ at least 20 types of tRNA in each cell

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12
Q

Describe the structure of tRNA.

What are the functions of its structural components?

A

4 arms form cloverleaf-like secondary structure

  • acceptor arm with posttranslationally added CCA 3’-terminal → specific AA binds here
  • TψC contains ψ = pseudouridine →binds to site of protein synth. on ribosome
  • anticodon loop → binds to rRNA
  • D arm often contains dihydrouridine → important for recognition of aminoacyl-tRNA synthetase
  • 5’ terminal phosphate group
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13
Q

What is the function of the CCA tail?

How is it special in eukaryotes?

A

AA specific for tRNA binds here

in eukaryotes added during processing of tRNA, hence not coded by tRNA gene

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14
Q

Some tRNA bases are often modified.

How are they modified and what is their function?

A

often by methylation (or deamidation)

→ affect the tRNA’s interaction with ribosomes

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15
Q

What is the function of aminoacyl-tRNAs?

They are synthesized by which enzyme?

A

tRNA carrying its specific “activated” AA attached to its terminal 3’-OH of CCA

formed by individiual aminoacyl-tRNA synthetases

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16
Q

What is the function of aminoacyl-tRNA synthetase?

A
  1. AA binds to synthetase, activated through the linkage of its -C group directly to AMP → 5’ aminoacyl AMP
    NOTE: driven by hydrolysis of ATP molecule that donates the AMP
  2. tRNA binds to synthetase, AMP-linked -C group on AA then transferred to a -OH group at the 3ʹ of the tRNA acceptor arm → forms aminoacyl-tRNA w/ activated ester linkage

NOTE: irreversible

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17
Q

List some features of the aminoacyl-tRNA synthetase.

A
  • one synthetase for each AA (one AA for each tRNA)
  • high fidelity (1 incorrect AA/104 - 105 reactions) due to proofreading mechanism
  • use identity elements on tRNA to distinguish similar tRNAs
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18
Q

Explain the proofreading mechanism of aminoacyl-tRNA synthetases.

A

select correct AA by a two-step mechanism

  1. correct AA has highest affinity for the active-site pocket of its synthetase
  2. adenylated AA tried to force into a second editing pocket in the enzyme → excludes correct AA, while allowing access by closely related AAs
    if matching: AA is removed from AMP (or from the tRNA itself if the aminoacyl-tRNA bond has already formed) by hydrolysis

= hydrolytic editing (analogous to the exonucleolytic proofreading by DNA polymerases)

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19
Q

What is wobbling?

A
  • first 2 base pairs of codon-anticodon are Watson-Crick base pairs
  • last base pair can wobble, hence form Hoogsteen base pair

→ main reason for degeneracy of genetic code

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20
Q

Which nucleotides on anticodon can wobble?

List pairs.

A
  • G also w/ U
  • U also w/ G
  • I w/ A, U, C
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21
Q

Where does translation and transcription happen in pro- and eukaryotes?

A
  • in prokaryotes: coupled,
    translation on free ribosomes and polyribosomes
  • in eukaryotes: compartmentalized, transcription in nucleus, translation on free cytoplasmic ribosomes and rER
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22
Q

Describe the general structure of a ribosome.

A

differences in number/size of their rRNA and protein components

  • small subunit: provides framework on which tRNAs are accurately matched to codons of mRNA
  • large subunit: catalyzes the formation of peptide bonds that link AAs together into a polypeptide chain

+ additionally proteins associated w/ subunits on surface

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23
Q

Describe the structure and weight of both ribosomal subunits in pro- and eukaryotes.

A

S refers to Svedberg units, describes weight

  • 70S ribosomes = prokaryotic ribosomes
    • large subunit has 23S and 5S rRNA
      → w/ proteins: 50S rRNA
    • small subunit has 16S
      → w/ proteins: 30S rRNA
  • 80S ribosomes = eukaryotic ribosomes
    • large subunit has 28S and 5S rRNA
      → w/ proteins: 60S rRNA
    • small subunit has 18S
      → w/ proteins: 40S rRNA

​→ eukaryotic ribosomes are heavier due to larger ribosomes + more associated proteins

24
Q

Where are eukaryotic ribosomes synthesized?

A

in the nucleolus

25
Q

Differentiate btw binding sites on ribosomes.

Function?

A

form complex w/ mRNA and tRNA, connect AAs at A site to the growing polypeptide

  • mRNA-binding site: on small subunit
  • E site: transiently holds “used” deacylated tRNA before it exits
  • P site: holds tRNA carrying growing polypeptide chain
  • A site: holds tRNA w/ next AA to be added
26
Q

In which direction does translation occur?

A

mRNA translated in 5’→3’ direction

NOTE: N-terminal end of a protein is made first, then AAs added one by one to the C-terminus of the polypeptide chain

27
Q

In which direction is the anticodon on tRNA read?

A

3’ → 5’
REMEMBER: left side of tRNA with short P-terminal is 3’, hence left base on anticodon read first

28
Q

Explain the general mechanism of translation.

What are the 3 basic steps?

A
  1. initiation: protein synthesis starting at AUG codon
  2. elongation: peptide bonds are formed btw 2 adjacent AAs, nascent polypeptide remains attached C-terminally via tRNA to ribosome
  3. termination: when stop codon reached, ester bonds btw protein and tRNA hydrolytically cleaved
29
Q

What is mono-, polycistronic mRNA?

Where do both types usually occur?

A
  • polycistronic: mRNA codes for more than protein
    → common for prokaryotic mRNAs
  • monocistronic: mRNA codes only for one peptide
    → common for eukaryotic mRNAs
30
Q

What are the main distinguishing features of initiation of translation in prokaryotes?

A
  • no preinitiation complexes
  • use Shine-Delgarno sequence
  • use fMet (formylmethionyl) instead of Met to bind to AUG start codon
  • require much less initiation factors (IFs)
    (in eukaryotes those are called eIF)
  • not regulated by phosphorylation
31
Q

Prokaryotic start codons are different than eukaryotic ones..

How?

A

have Shine-Delgarno sequence upstream of AUG start codon on mRNA

  • untranslated 3 - 9bp sequence
  • binds to small subunit (16S rRNA)

NOTE: ONLY in prokaryotes

32
Q

Explain the stepwise process of prokaryotic initiation.

A
  1. IF-3 and IF-1 bind to small subunit
  2. small subunit associates w/ mRNA
  3. GTP-IF-2 binds to P site of small subunit, then pairs fMet-tRNA to AUG codon on mRNA
  4. large subunit associates, hydrolyzes GTP to GDP + Pi
  5. all IFs are released, complete 70S initiation complex is formed
33
Q

What are the 3 steps of the elongation of translation?

A
  1. binding of aminoacyl-tRNA to A site
  2. peptide bond formation
  3. translocation + expulsion of deacylated tRNA from P- and E-sites
34
Q

Elongation can be divided into 3 basic steps..

What happens in the 1st step in prokaryotes, after the 70S complex is formed?

A

binding of aminoacyl-tRNA to A site

  1. ​​complex of EF-Tu-GTP, aminoacyl-tRNA, bind to A site, EF-Tu checks if correct aminoacyl-tRNA bound
  2. when correct tRNA bound, EF-Tu hydrolzes GTP → EF-Tu-GDP and Pi released
35
Q

What is the function of EF-Ts?

A

GEF protein

releases GDP from EF-Tu, rebinds GTP for new elongation

36
Q

Elongation can be divided into 3 basic steps..

What happens in the 2nd step in prokaryotes, after 2 aminoacyl-tRNAs are bound to the ribosome?

A

peptide bond formation
catalyzed by adenine in peptidyl transferase center of large ribosomal subunit

nucleophilic attack of C-terminal of bound peptidyl-tRNA on P site by ⍺-N group of aminoacyl-tRNA on A site

⇒ attachment of nascent peptide chain to A site, deacetylated tRNA remains in P site

37
Q

Elongation can be divided into 3 basic steps..

What happens in the last step in prokaryotes, after formation of a new peptide bond?

A

translocation + expulsion

  1. EF-G-GTP binds
  2. hydrolysis of GTP causes translocation of ribosome by one codon → deacylated tRNA now on E site, peptidyl-tRNA on P site, A site empty again
  3. EF-G-GDP released, deacetylated tRNA leaves ribosome
38
Q

How is eukaryotic elongation different from prokaryotic?

A

only different translation factors used

  • eEF1A instead of EF-Tu (association of aminoacyl-tRNA w/ ribosome)
  • eEF1B instead of EF-Ts (GEF)
  • eEF2 instead of EF-G (translocation)
39
Q

How is translation terminated in prokaryotes?

A
  1. when stop codon reached, RF associates instead of new aminoacyl-tRNA at A site
    • RF-1 for UAA, UAG
    • RF-2 for UAA, UGA
  2. RF-3-GTP also binds, hydrolzyes its GTP
  3. RF-1 or 2 now hydrolyzes peptidyl-tRNA, releasing the peptide

→ ribosome dissociates into subunits, mRNA, tRNA also released

40
Q

How is termination in eukaryotes different from that in prokaryotes?

A

only different translation factors used

  • eRF1 instead of RF-1 or RF-2: recognizes all 3 stop codons
  • eRF3 instead of RF-3 (hydrolysis of GTP)
  • ribosomal subunits dissociate when new eIFs bind
41
Q

How come, eRF1 (or RF-1/2) able to bind to the mRNA?

A

due to its structural similarity to tRNA

= molecular mimicry

42
Q

Eukaryotic initiation is somewhat different from that in prokaryotes.

What are the 4 general steps of the initiation here?

A
  1. formation of 43S preinitiation complex
  2. formation of 48S preinitiation complex
  3. scanning of mRNA
  4. formation of 80S initiation complex​
43
Q

Eukaryotic initiation can be divided into 4 basic steps..

What happens in the 1st step?

A

formation of 43S preinitiation complex

  1. eIF1, eIF1A and eIF3 bind to small subunit
  2. complex of eIF2, Met-tRNA and GTP bind to P-site
44
Q

Eukaryotic initiation can be divided into 4 basic steps..

What happens in the 2nd step, after the 43S preinitiation complex has formed?

A

formation of 48S preinitiation complex

multienzyme complex eIF4 binds to 5’ cap of mRNA and to poly-A binding protein I (PABP) which is associated w/ 3’ end of poly A tail

→ entire mRNA forms a closed loop, ensuring that only intact mRNA is translated

45
Q

Eukaryotic initiation can be divided into 4 basic steps..

What happens in the 3rd step, after the 48S preinitiation complex has formed?

A

scanning

eIF4 has helicase activity, starts unwinding mRNA in 5’→3’ until it finds AUG start codon/Kozak sequence

→ when found, Met binds to mRNA, GTP hydrolyzed, eIF2-GDP leaves 48S preinitiation complex

46
Q

What is the Kozak sequence?

A

typical sequence in eukaryotes surrounding the AUG start codon to intiate translation

47
Q

Which factor has the GEF function in eukaryotes to restore eIF2-GTP

A

eIF2B

48
Q

Eukaryotic initiation can be divided into 4 basic steps..

What happens in the 4th step, after the the 48S preinitiation has bind Met to the mRNA?

A

formation of 80S initiation complex

  1. eIF5-GTP and eIF1A associate w/ small ribosomal subunit
  2. large ribsomal subunit can now bind to 48S preininitiation complex due to eIF5, release of eIF1 and eIF3
  3. large ribosomal subunit hydrolyzes eIF5-GTP, causing it to leave as well

→ 80S initiation complex is ready for elongation

49
Q

How much energy is required to add 1 AA to the nascent polypeptide chain?

A

4 macroergic bonds (BUT: 1 ATP, 2 GTP)

  1. 1 ATP to AMP needed for charging of tRNA
  2. 1 GTP needed for binding of aminoacyl-tRNA on A site
  3. 1 GTP needed for translocation of ribosome
50
Q

Only the translation of eukaryotes is regulated.

What are the 2 mechanisms?

A

eIF2 and eIF4 not present in prokaryotes

  • eIF2 can be phosphorylated, causing inhibition of translation
  • eIF4 can be phosphorylated, prerequisite for translation
51
Q

What is the mechanism behind eIF2 phosphorylation?

A

eIF2 needs bound GTP to be active

can be phosphorylated at α-subunit, then eIF2B (GEF) cannot be released to exchange GTP w GDP → remains inactivated

52
Q

Which 4 enzymes are able to phosphorylate eIF2, hence inactivating translation?

A
  • HRI (heme regulated inhibitor):
    present in erythrocyte precursors, activated in response to heme deficit to switch off translation of globin mRNA as long as deficit persists
  • GCN2 (general AA control):
    activated in response to free tRNAs, as a signal when cell is lacking AAs for translation
  • PERK (pancreatic kinase of ER lumen)
    activated when unfolded proteins in ER accumulate
  • PKR (RNA dependent protein kinase)
    activated in response to binding of dsRNA, as can be found in some viral infections
53
Q

Explain how eIF4 contributes to the regulation of translation in eukaryotes.

A

eIF4 has multiple subunits

  • eIF4E binds 5’cap and eIF4G
    • cannot bind cap if associated w/ eIF4-BP (binding protein)
    • only binds eIF4-BP if NOT phosphorylated
    • eIF4-BP is phosphorylated by mTOR
  • ​eIF4G binds to small ribosomal subunit
54
Q

Which signals activate mTOR, which inhibit it?

A

mTOR (mammalian target of rapamycin)

  • activated in response to GFs, accumulating AAs
  • inhibited by hypoxia, AMP and rapamycin
55
Q

How do some viruses trigger the translation of their mRNA once they infected the cell?

A

initiate their translation independent of 5’ cap

  1. cleave eIF4G, suppressing translation of host’s mRNA → creates a C-terminal on eIF4G
  2. small ribosomal subunit, containing cleaved eIF4G now binds to IRES (internal ribosome entry site) on viral mRNA
56
Q

List some viruses that have specific IRES, hence are able to translate their mRNA in a cap indpendent manner.

A
  • picornaviruses (ex: poliovirus)
  • hepatitis c virus
  • encephalomyocarditis virus (EMCV)
  • cricket paralysis virus (CrPV)
57
Q

List some antibiotics and their effect on translation.

A

inhibit translation

Biochemistry I (13 decks)

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