Structural and functional properties of ribosomes crosslinked with dimethylsuberimidate

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• The 70S ribosome is the primary machinery for protein synthesis in prokaryotes.
• The role of the 70S ribosome and its subunits in initiating protein synthesis remains a key question.

Purpose of the Study:

• To investigate whether a 30S ribosomal subunit-formylmethionyl-tRNA-mRNA complex is a mandatory intermediate in protein synthesis.
• To determine if 70S ribosomes must dissociate into subunits to initiate translation with natural mRNAs.

Main Methods:

• Crosslinking of 70S ribosomes from Escherichia coli using dimethylsuberimidate.
• Treatment of crosslinked ribosomes with high salt concentrations to generate distinct ribosomal particles.
• Assays for ribosomal subunit binding, N-acetylphenylalanyl-tRNA binding, and polyphenylalanine synthesis.
• Testing protein synthesis activity with bacteriophage f2 RNA as messenger RNA.

Main Results:

• Crosslinked 70S ribosomes, unable to dissociate, retained 80% of their activity for polyphenylalanine synthesis with poly(U) mRNA.
• Crosslinked ribosomes showed reduced binding of formylmethionyl-tRNA (fMet-tRNA).
• Crosslinked ribosomes were inactive in protein synthesis using bacteriophage f2 RNA, indicating a failure in initiation with natural mRNAs.

Conclusions:

• 70S ribosomes must dissociate into their 30S and 50S subunits to initiate protein synthesis with natural messenger RNAs.
• The formation of a 30S subunit-formylmethionyl-tRNA-mRNA complex is essential for initiating translation with natural mRNAs.