Genetic code + translation

Question 1

What are pri-miRNAs?

What is their function?

Answer 1

primary transcripts miRNAs transcribed by RNA pol II

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

Question 2

What is a codon?

How many are there?

Answer 2

three letter base code w/ 64 possibilities

→ coding for 20 different amino acids

Question 3

List the 5 features of the genetic code.

Answer 3

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

DUNo NpU

Question 4

The genetic code is non-punctuated.

What does it mean?

Answer 4

translated continuously until stop codon reached, no bases skipped

Question 5

The genetic code is non-overlapping.

What does it mean?

Answer 5

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

Question 6

The genetic code is degenerate.

What does it mean?

Answer 6

18 of 20 AAs coded by more than 1 codon

only exceptions: Met and Trp

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

Question 7

The genetic code is almost universal.

What does it mean?

Answer 7

most codons code for the same AA in different organism

BUT: not true for mitochondria

Question 8

The genetic code is unambiguous.

What does it mean?

Answer 8

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

Question 9

Which codons code start and stop signals for translation?

Answer 9

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

Question 10

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

Which amino acids and how are they translated?

Answer 10

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

Question 11

What is the function of tRNA?

Answer 11

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

Question 12

Describe the structure of tRNA.

What are the functions of its structural components?

Answer 12

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

Question 13

What is the function of the CCA tail?

How is it special in eukaryotes?

Answer 13

AA specific for tRNA binds here

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

Question 14

Some tRNA bases are often modified.

How are they modified and what is their function?

Answer 14

often by methylation (or deamidation)

→ affect the tRNA’s interaction with ribosomes

Question 15

What is the function of aminoacyl-tRNAs?

They are synthesized by which enzyme?

Answer 15

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

formed by individiual aminoacyl-tRNA synthetases

Question 16

What is the function of aminoacyl-tRNA synthetase?

Answer 16

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

Question 17

List some features of the aminoacyl-tRNA synthetase.

Answer 17

• 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

Question 18

Explain the proofreading mechanism of aminoacyl-tRNA synthetases.

Answer 18

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)

Question 19

What is wobbling?

Answer 19

• 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

Question 20

Which nucleotides on anticodon can wobble?

List pairs.

Answer 20

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

Question 21

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

Answer 21

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

Question 22

Describe the general structure of a ribosome.

Answer 22

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

Question 23

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

Answer 23

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

Question 24

Where are eukaryotic ribosomes synthesized?

Answer 24

in the nucleolus

Question 25

Differentiate btw binding sites on ribosomes. Function?

Answer 25

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 **_e_**xits * **P site:** holds tRNA carrying growing **_p_**olypeptide chain * **A site:** holds tRNA w/ next **_A_**A to be added

Question 26

In which direction does translation occur?

Answer 26

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

Question 27

In which direction is the anticodon on tRNA read?

Answer 27

**3' → 5'** _{REMEMBER: left side of tRNA with short P-terminal is 3', hence left base on anticodon read first}

Question 28

Explain the general mechanism of translation. What are the 3 basic steps?

Answer 28

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

Question 29

What is mono-, polycistronic mRNA? Where do both types usually occur?

Answer 29

* **polycistronic:** mRNA codes for more than protein → common for _prokaryotic_ mRNAs * **monocistronic:** mRNA codes only for one peptide → common for _eukaryotic_ mRNAs

Question 30

What are the _main distinguishing_ features of initiation of translation in prokaryotes?

Answer 30

* **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**

Question 31

Prokaryotic start codons are different than eukaryotic ones.. How?

Answer 31

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

Question 32

Explain the stepwise process of prokaryotic initiation.

Answer 32

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 + P_i** 5. all **IFs are released**, complete _70S initiation complex_ is formed

Question 33

What are the 3 steps of the elongation of translation?

Answer 33

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

Question 34

Elongation can be divided into 3 basic steps.. What happens in the 1^st step _in prokaryotes_, after the 70S complex is formed?

Answer 34

_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 P_i released

Question 35

What is the function of EF-Ts?

Answer 35

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

Question 36

Elongation can be divided into 3 basic steps.. What happens in the 2^nd step _in prokaryotes_, after 2 aminoacyl-tRNAs are bound to the ribosome?

Answer 36

_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

Question 37

Elongation can be divided into 3 basic steps.. What happens in the last step _in prokaryotes_, after formation of a new peptide bond?

Answer 37

_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

Question 38

How is eukaryotic elongation different from prokaryotic?

Answer 38

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)

Question 39

How is translation terminated in prokaryotes?

Answer 39

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

Question 40

How is termination in eukaryotes different from that in prokaryotes?

Answer 40

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_

Question 41

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

Answer 41

due to its structural similarity to tRNA = **molecular mimicry**

Question 42

Eukaryotic initiation is somewhat different from that in prokaryotes. What are the 4 general steps of the initiation here?

Answer 42

1. formation of **43S **_pre_**initiation complex** 2. formation of **48S **_pre_**initiation complex** 3. **scanning** of mRNA 4. formation of **80S initiation complex**​

Question 43

Eukaryotic initiation can be divided into 4 basic steps.. What happens in the 1^st step?

Answer 43

_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

Question 44

Eukaryotic initiation can be divided into 4 basic steps.. What happens in the 2^nd step, after the 43S preinitiation complex has formed?

Answer 44

_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

Question 45

Eukaryotic initiation can be divided into 4 basic steps.. What happens in the 3^rd step, after the 48S preinitiation complex has formed?

Answer 45

_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

Question 46

What is the Kozak sequence?

Answer 46

typical sequence in _eukaryotes_ **surrounding the AUG start codon** to intiate translation

Question 47

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

Answer 47

**eIF2B**

Question 48

Eukaryotic initiation can be divided into 4 basic steps.. What happens in the 4^th step, after the the 48S preinitiation has bind Met to the mRNA?

Answer 48

_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

Question 49

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

Answer 49

**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

Question 50

Only the translation of eukaryotes is regulated. What are the 2 mechanisms?

Answer 50

eIF2 and eIF4 not present in prokaryotes * **eIF2** can be **phosphorylated**, causing _inhibition of translation_ * **eIF4** can be **phosphorylated**, _prerequisite for translation_

Question 51

What is the mechanism behind eIF2 phosphorylation?

Answer 51

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_

Question 52

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

Answer 52

* _**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

Question 53

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

Answer 53

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

Question 54

Which signals activate mTOR, which inhibit it?

Answer 54

mTOR (mammalian target of rapamycin) * _activated_ in response to **GFs**, **accumulating AAs** * _inhibited_ by **hypoxia, AMP and rapamycin**

Question 55

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

Answer 55

_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

Question 56

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

Answer 56

* **picornaviruses** (ex: poliovirus) * **hepatitis c virus** * **encephalomyocarditis virus** (_EMCV_) * **cricket paralysis virus** (_CrPV_)

Question 57

List some antibiotics and their effect on translation.

Answer 57

inhibit translation