Translation-Protein manufactory Miguel Suárez Barrera- Microbiólogo Industrial MSc

STRUCTURE AND SUBUNITS Ribosomes are large ribonucleoprotein particles that contain more RNA than protein and dissociate into large and small subunits. Electron microscopic images of bacterial ribosomes and subunits reveal their shapes. Photographs kindly provided by James Lake.

THE STAGE OF PROTEIN SYNTHESIS Size comparisons show that the ribosome is large enough to bind tRNAs and mRNA. The ribosome has two sites for binding charged tRNA.

INITIATION IN BACTERIA NEEDS 30S SUBUNITS AND ACCESSORY FACTORS Initiation requires 30S subunits that carry IF-3. Initiation requires free ribosome subunits. When ribosomes are released at termination, they dissociate to generate free subunits. Initiation factors are present only on dissociated 30S subunits. When subunits reaassociate to give a functional ribosome at initiation, they release the factors.

A SPECIAL INITIATOR TRNA starts the polypeptide chain The initiator N-formyl- methionyl- tRNA (fMet- tRNAf) is generated by formylation of methionyl- tRNA, using formyl- tetrahydrofol ate as cofactor. Only fMet-tRNAf can be used for initiation by 30S subunits; only other aminoacyl- tRNAs (aa-tRNA) can be used for elongation by 70S ribosomes fMet-tRNAf has unique features that distinguish it as the initiator tRNA. IF-2 is needed to bind fMet-tRNAf to the 30S-mRNA complex. After 50S binding, all IF factors are released and GTP is cleaved.

INITIATION INVOLVES BASE PAIRING BETWEEN mRNA and rRNA Figure 6.15 Ribosome-binding sites on mRNA can be recovered from initiation complexes. Figure 6.16 Initiation occurs independently at each cistron in a polycistronic mRNA. When the intercistronic region is longer than the span of the ribosome, dissociation at the termination site is followed by independent reinitiation at the next cistron.

ELONGATION FACTOR T LOADS AMINOACYL-tRNA into the A-side EF-Tu-GTP places aminoacyl-tRNA on the ribosome and then is released as EF-Tu-GDP. EF- Ts is required to mediate the replacement of GDP by GTP. The reaction consumes GTP and releases GDP. The only aminoacyl-tRNA that cannot be recognized by EF-Tu-GTP is fMet- tRNAf, whose failure to bind prevents it from responding to internal AUG or GUG codons.

TRANSLOCATION MOVES THE RIBOSOME

THREE CODONS TERMINATE PROTEIN SYNTHESIS Molecular mimicry enables the elongation factor Tu-tRNA complex, the translocation factor EF- G, and the release factors RF1/2-RF3 to bind to the same ribosomal site. The RF (release factor) terminates protein synthesis by releasing the protein chain. The RRF (ribosome recycling factor) releases the last tRNA, and EF-G releases RRF, causing the ribosome to dissocuate.