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 12. Beskrive kort initiering af translation ud fra følgende:
Stryer, s. 827-9
 Stryer, s.833, fig. 29.27
Stryer, s.837
Devlin, s.245-7

 

Prokaryote celler:

Ribosom positioneringssekvens (Shine-Delgarno)

The Shine-Delgarno sequence is a purine-rich sequence (GAGGGG) found in the initiator region of prokaryotic mRNA. It is located about 10 nucleotides upstream the initiator codon AUG.

The Shine-Delgarno sequence binds to a complementary region near the 3’-end of the 16S rRNA (small subunit of the ribosome). The number of base pairs between these mRNA and rRNA ranges from three to nine.

This region therefore has influence on where the translation process starts. Protein synthesis actually begins with the interaction of the Shine-Delgarno sequence of the mRNA with the rRNA of the ribosome.


AUG startkodon i mRNA


Another region, which of course has influence on where the translation starts is the initiation sequence AUG. Prokaryotic mRNA is polycistronic, meaning it encodes for several polypeptide chains, so it has several AUG starting sequences. The very first AUG sequence is always located more then 25 nucleotides from the 5’-end of the mRNA.

When the complex between the Shine-Delgarno sequence and the rRNA of the small subunit is formed, the initiator fMet-tRNA (charged with formylmethionine) binds to the initiator AUG sequence through Watson-Crick complementary base pairing.

Important: Shine-Delgarno of mRNA binds to rRNA, while AUG of mRNA binds to tRNA.


30S ribosomal subunit
 

The 30S ribosomal subunit, the small subunit, contains a 16S rRNA that has a pyrimidine rich sequence towards its 3’-end, which binds complementary to the purine-rich Shine-Delgarno sequence and helps position the mRNA in the right way. So, the codon-anticodon interaction between mRNA and tRNA happens in the small subunit.

The 30S ribosomal unit forms a complex with IF1 and IF3, which prevents premature joining between the large and small subunit, creating a dead-end ribosome.

The 30S subunit has an A, P and E site. It is in the P-site that the tRNA and mRNA make the codon-anticodon interaction. The A site is free for the next tRNA complementary to the second codon in the sequence.

 


Initieringsfaktorer (tre proteiner)
 

mRNA and fMet-tRNA must be brought to the ribosome for protein synthesis to begin.

There are three proteins, called initiation factors, IF1, IF2 and IF3 that are essential for the translation.  

IF1 and IF3 bind to the small subunit of the ribosome and prevent it from premature rejoining with the large subunit.

IF2 binds GTP, and the conformational change introduced in IF2 enables it to bind with fMet-tRNA.

The IF2-fMet-tRNA-GTP complex can then bind to mRNA, correctly positioned due to the Shine-Delgarno sequence with IF1-IF3-16srRNA, and the 30S initiation complex is formed.
Stryer, s.833 - fig. 29.27


fMet-tRNA (i P-sitet)
Stryer, s.828 - fig.29.21

Prokaryotic translation is initiated by a formyl-methionine tRNA. The initiator tRNA is first charged with methionine by aminoacyl-tRNA synthetase. The methionyl-tRNA is then formylated by transformylase.

 The fMet-tRNA is recognized by IF2, which in turn has to be bound to GTP in order to bind the small subunit of the ribosome. The tRNA is located in the P-site, since it is the donor tRNA, and it will give its amino acid to the growing chain.   

 The anticodon loop of tRNA complementary binds to the codon sequence of mRNA, after mRNA has been positioned rightly by the Shine-Delgado sequence and 16SrRNA.

Other methionines that have to be incorporated in the growing polypeptide chain are not formylated.

30S initiation complex:

  1. mRNAs Shine-Delgado sequence binds complementary to the 16S rRNA of the 30S subunit

  2. IF1 and IF3 bind to the 30S subunit and prevent it from prematurely joining the 50S subunit

  3. IF2 binds GTP and after that binds the fMet-tRNA

  4. fMet-tRNAs anticodon sequence recognises AUG sequence in the mRNA

 


Eukaryote celler:

Første AUG fra 5´-CAP enden af mRNA er startkodon

 

Eukaryotic mRNA is monocistronic, meaning it only encodes for one polypeptide chain. That means that there is only one initiation sequence, which here also is AUG.

It is usually the first AUG encountered in mRNA from its 5’-end that is the initiation sequence, but sometimes the surrounding nucleotide sequence or secondary structure is not appropriate for initiation and a later AUG is then selected.

The AUG codon is recognized by the anticodon sequence of the initiating met-tRNA.


40S ribosomal subunit
Stryer, s. 837 - fig.29.33
 

The 40S unit is contacted by an initiation factor, bound to GTP and Met-tRNA i. The initiation factor prevents the 40S ribosomal subunit from binding with the large subunit.

This complex then attaches to the cap of the 5’-end of mRNA and searches for the AUG initiation sequence, so there is no need for a Shine-Delgarno sequence. The scanning process of mRNA utilises ATP.  

When the anticodon of Met-tRNA is paired with the AUG codon, the target has been found. 

The 40S subunit has an A, P and E site. It is in the P-site that the tRNA and mRNA make the codon-anticodon interaction. The A site is free for the next tRNA complementary to the second codon in the sequence.
 

 
Initierings faktorer (mange proteiner)
 

Eukaryotes initialise many more initiation factors then prokaryotes and their interplay are more complicated. The proteins are called eIF: eIF1, eIF2 anf eIF3.   


Met-tRNA (i P-sitet)
  

The initiation tRNA in eukaryotes is also Met-tRNA i, but it isn’t formylated. No other tRNA can replace it in the initiation complex, not even the Met-tRNAe, which carries a methionine which is to be incorporated during elongation of the polypeptide chain.

The initiation tRNA is bounded by an initiation factor-GTP complex, and the associated with the 40S subunit which then scans the mRNA.   

As usually, the anticodon of tRNA recognizes the AUG codon of mRNA.

Initiation in eukaryotes:

  1. an initiation factor binds to ATP, binds to Met-tRNA i

  2. they bind to the 40S subunit

  3. the complex scans for the AUG

  4. complementary pairing between tRNA anticodon and mRNA codon

 

The basic difference in the initiation between prokaryotes and eukaryotes is that eukaryotes do not have a Shine-Delgarno sequence, so the ribosome has to scan the mRNA instead of using ribosome positioning. Prokaryotes have polycistronic mRNA, meaning more Shine-Delgarno sequences, more AUG start codons and can not be scanned.
 

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