Shine-Dalgarno sequences

The Shine-Salgano interaction is a base pairing interaction that occurs during translation initiation in prokaryotes between the Shine-Dalgarno sequence on messenger RNA (mRNA) and the anti-Shine-Dalgamo... 

Translation Initiation 305 subrmit binds to Shine-Dalgarno sequence on mRNA j 40S subunit assodates with 5 cap on mRNA... 

Vectors for recombinant DNA and cloning Plasmid pBR322 Restriction site Replication origin Resistance to antibiotic(s) Expression vector also requires Promoter Shine-Dalgarno sequence Other vectors phage, YACs... 

Answer C. Incorporation of a Shine-Dalgarno sequence into the expression vector will promote ribosome binding to the translation start site on the mRNA produced by transcription of the cDNA insert. 

The pTDl vector should not be used as the expression vector for an E. coli cell-free protein synthesis system because this vector does not contain Shine-Dalgarno sequence. 

As opposed to the case in procaryotes, eucaryotic translation does not require a specific sequence for the binding of the ribosome. Procaryotes rely on the Shine-Dalgarno sequence, which is complementary to sequences of the 16S RNA of the 308 subunit. The Shine-Dalgamo sequence mediates the binding of mRNA to the 308 ribosome and ensures the correct positioning of the AUG initiation codon. 

Initiation The components of the translation system are assembled, and mRNA associates with the small ribosomal subunit. The process requires initiation factors. In prokaryotes,a purine-rich region (the Shine-Dalgarno sequence) of the mRNA base-pairs with a complementary sequence on 16S rRNA, resulting in the positioning of the mRNA so that translation can begin. The 5 -cap on eukaryotic mRNA is used to position that structure on the ribosome. The initiation codon is 5 -AUG-3. ... 

The Sequence of the 3 End of E. coli 16S RNA and Some Shine-Dalgarno Sequences at the 5 End of Bacterial mRNAs... 

The pyrimidine-rich complement to the Shine-Dalgarno sequence... 

The importance of the Shine-Dalgarno sequence is underscored by the action of the bacterial toxin colecin E3. This toxin inactivates the small subunit of the prokaryotic ribosome by cleavage of about 50 residues from the 3 terminus of 16S rRNA. The cleavage disrupts the sequence that is complementary to the Shine-Dalgarno sequence and thus specifically inhibits the initiation process. Because of the fundamental differences between prokaryotic and eukaryotic initiation just described, colecin E3 does not inhibit the eukaryotic ribosome. 

E. coli has three initiation factors (fig. 29.13) bound to a small pool of 30S ribosomal subunits. One of these factors, IF-3, serves to hold the 30S and 50S subunits apart after termination of a previous round of protein synthesis. The other two factors IF-1 and IF-2, promote the binding of both fMet-tRNA,Mel and mRNA to the 30S subunit. As we noted before, the binding of mRNA occurs so that its Shine-Dalgarno sequence pairs with 16S RNA and the initiating AUG sequence with the anticodon of the initiator tRNA. The 30S subunit and its associated factors can bind fMet-tRNAjMet and mRNA in either order. Once these ligands are found, IF-3 dissociates from the 30S, permitting the 50S to join the complex. This releases the remaining initiation factors and hydrolyzes the GTP that is bound to IF-2. The initiation step in prokaryotes requires the hydrolysis of one equivalent of GTP to GDP and Pj. 

A segment of the A genome containing the Prm and nearby PL, PR, and PRb. promoters. Transcripts that can originate from the three promoters are shown by the wavy lines. The PRe and -PRM require different activators for expression. The transcript from PRF is 10 times more effective in cl expression because it has a Shine-Dalgarno sequence for ribosome attachment. 

The first codon translated in all mRNAs is AUG which codes for methionine. This AUG is called the start codon or initiation codon. Naturally, other AUG codons also occur internally in an mRNA where they encode methionine residues internal to the protein. Two different tRNAs are used for these two types of AUG codon tRNAfMet is used for the initiation codon and is called the initiator tRNA whereas tRNAmMet is used for internal AUG codons. In prokaryotes the first amino acid of a new protein is /V-formylmethionine (abbreviated fMet). Hence the aminoacyl-tRNA used in initiation is fMet-tRNAfMet. It is essential that the correct AUG is used as the initiation codon since this sets the correct reading frame for translation (see Topic HI). A short sequence rich in purines (5 -AGGAGGU-3 ), called the Shine-Dalgarno sequence, lies 5 to the AUG initiation codon (Fig. 3) and is complementary to part of the 16S rRNA in the small ribo-somal subunit. Therefore this is the binding site for the 30S ribosomal subunit... 

Shine-Dalgarno sequence (ribosome binding site) Initiation codon... 

The small subunit then binds to the mRNA via the Shine-Dalgarno sequence and moves 3 along the mRNA until it locates the AUG initiation codon. 

Eukaryotic ribosomes are larger (80S) and more complex than prokaryotic ribosomes (70S). Initiation is basically similar in prokaryotes and eukaryotes except that in eukaryotes at least nine initiation factors are involved (cf. three factors in prokaryotes), the initiating amino acid is methionine (cf. N-formylmethionine in prokaryotes), eukaryotic mRNAs do not contain Shine-Dalgarno sequences (so the AUG initiation codon is detected by the ribosome scanning instead), and eukaryotic mRNA is monocistronic (cf. some polycistronic mRNAs in prokaryotes). Initiation in eukaryotes involves the formation of a 48S preinitiation complex between the 40S ribosomal subunit, mRNA, initiation factors and Met-tRNA 61. The ribosome then scans the mRNA to locate the AUG initiation codon. The 60S ribosomal subunit now binds to form the 80S initation complex. 

The ribosome has a problem similar to that of RNA polymerase the ribosome must find the point in the mRNA at which to begin translation. In prokaryotes the site is marked by a tract called the Shine-Dalgarno sequence, about ten nucleotides upstream from the initiation site. Initiation occurs at the first subsequent AUG sequence. (AUG codes for the amino acid methionine.) In eukaryotes, initiation usually begins simply at the first AUG sequence from the 5 -end of the mRNA. 

The role of this purine-rich region, called the Shine-Dalgarno sequence, became evident when the sequence of 16S... 

Fig. 6. Distance from the Shine-Dalgarno sequence to the Initiation Codon in archaeal genes. Data for this graph were obtained from refs. [8,9,12,84,114,120,122,123,133,140,142-146,148-152].

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