1. Protein synthesis  30.4 Ribosome Structure and Assembly  30.5 Mechanics of Protein Synthesis  30.4 Ribosome Structure and Assembly  30.5 Mechanics of Protein Synthesis

2. 30.4 Ribosome Structure and Assembly  E. coli ribosome is 25 nm diameter, 2520 kD in mass, and consists of two unequal subunits that dissociate at < 1mM Mg 2+  30S subunit is 930 kD with 21 proteins and a 16S rRNA  50S subunit is 1590 kD with 31 proteins and two rRNAs: 23S rRNA and 5S rRNA  These ribosomes and others are roughly 2/3 RNA  20,000 ribosomes in a cell, 20% of cell's mass  E. coli ribosome is 25 nm diameter, 2520 kD in mass, and consists of two unequal subunits that dissociate at < 1mM Mg 2+  30S subunit is 930 kD with 21 proteins and a 16S rRNA  50S subunit is 1590 kD with 31 proteins and two rRNAs: 23S rRNA and 5S rRNA  These ribosomes and others are roughly 2/3 RNA  20,000 ribosomes in a cell, 20% of cell's mass

4. Ribosomal RNA  3 rRNA molecules  23S, 16S, 5S  Derived from a single 30S rRNA precursor transcript  Extensive intrachain H-bonding  2/3 rRNA is helical  3 rRNA molecules  23S, 16S, 5S  Derived from a single 30S rRNA precursor transcript  Extensive intrachain H-bonding  2/3 rRNA is helical

5. Ribosomal Proteins  One of each per ribosome, except L7/L12 with 4  L7/L12 identical except for extent of acetylation at N-terminus  Only one protein is common to large and small subunits: S20 = L26  Variety of structures, still being characterized  One of each per ribosome, except L7/L12 with 4  L7/L12 identical except for extent of acetylation at N-terminus  Only one protein is common to large and small subunits: S20 = L26  Variety of structures, still being characterized

6. Ribosome Assembly/Structure  If individual proteins and rRNAs are mixed, functional ribosomes will assemble  Gross structures of large and small subunits are known - see Figure 30.12  A tunnel runs through the large subunit  Growing peptide chain is thought to thread through the tunnel during protein synthesis  If individual proteins and rRNAs are mixed, functional ribosomes will assemble  Gross structures of large and small subunits are known - see Figure 30.12  A tunnel runs through the large subunit  Growing peptide chain is thought to thread through the tunnel during protein synthesis

9. Eukaryotic Ribosomes  Mitochondrial and chloroplast ribosomes are quite similar to prokaryotic ribosomes, reflecting their supposed prokaryotic origin  Cytoplasmic ribosomes are larger and more complex, but many of the structural and functional properties are similar  See Table 30.6 for properties  Mitochondrial and chloroplast ribosomes are quite similar to prokaryotic ribosomes, reflecting their supposed prokaryotic origin  Cytoplasmic ribosomes are larger and more complex, but many of the structural and functional properties are similar  See Table 30.6 for properties

10. 30.5 Mechanics of Protein Synthesis  All protein synthesis involves three phases: initiation, elongation, termination  Initiation involves binding of mRNA and initiator aminoacyl-tRNA to small subunit, followed by binding of large subunit  Elongation: synthesis of all peptide bonds - with tRNAs bound to acceptor (A) and peptidyl (P) sites. See Figure 30.13  Termination occurs when "stop codon" reached  All protein synthesis involves three phases: initiation, elongation, termination  Initiation involves binding of mRNA and initiator aminoacyl-tRNA to small subunit, followed by binding of large subunit  Elongation: synthesis of all peptide bonds - with tRNAs bound to acceptor (A) and peptidyl (P) sites. See Figure 30.13  Termination occurs when "stop codon" reached

13. Prokaryotic Initiation  The initiator tRNA is one with a formylated methionine: f-Met-tRNA f Met  It is only used for initiation, and regular Met- tRNA m Met is used instead for Met addition  N-formyl methionine is first aa of all E.coli proteins, but this is cleaved in about half  A formyl transferase adds the formyl group (see Figure 30.15)  The initiator tRNA is one with a formylated methionine: f-Met-tRNA f Met  It is only used for initiation, and regular Met- tRNA m Met is used instead for Met addition  N-formyl methionine is first aa of all E.coli proteins, but this is cleaved in about half  A formyl transferase adds the formyl group (see Figure 30.15)

15. More Initiation  Correct registration of mRNA on ribosome requires alignment of a pyrimidine-rich sequence on 3'-end of 16S RNA with a purine-rich part of 5'-end of mRNA  The purine-rich segment - the ribosome-binding site - is known as the Shine-Dalgarno sequence (see Figure 30.17)  Initiation factor proteins, GTP, N-formyl-Met- tRNAfMet, mRNA and 30S ribosome form the 30S initiation complex  Correct registration of mRNA on ribosome requires alignment of a pyrimidine-rich sequence on 3'-end of 16S RNA with a purine-rich part of 5'-end of mRNA  The purine-rich segment - the ribosome-binding site - is known as the Shine-Dalgarno sequence (see Figure 30.17)  Initiation factor proteins, GTP, N-formyl-Met- tRNAfMet, mRNA and 30S ribosome form the 30S initiation complex

17. Events of Initiation  30S subunit with IF-1 and IF-3 binds mRNA, IF-2, GTP and f-Met-tRNA f Met (Figure 30.18)  IF-2 delivers the initiator tRNA in a GTP- dependent process  Loss of the initiation factors leads to binding of 50S subunit  Note that the "acceptor site" is now poised to accept an incoming aminoacyl-tRNA  30S subunit with IF-1 and IF-3 binds mRNA, IF-2, GTP and f-Met-tRNA f Met (Figure 30.18)  IF-2 delivers the initiator tRNA in a GTP- dependent process  Loss of the initiation factors leads to binding of 50S subunit  Note that the "acceptor site" is now poised to accept an incoming aminoacyl-tRNA

20. The Elongation Cycle  Elongation factor Tu will bring each aa-tRNA into the A site  Decoding center of 16S rRNA makes sure the proper aa tRNA is in the A site by direct surveillance  Peptide bond formation occurs by direct transfer of the peptidyl chain from the tRNA bearing it to the NH2 group of the new amino acid  Translocation of the one-residue-longer peptidyl tRNA to the P site to make room for the next incoming aa- tRNA at the A site.  Elongation factor Tu will bring each aa-tRNA into the A site  Decoding center of 16S rRNA makes sure the proper aa tRNA is in the A site by direct surveillance  Peptide bond formation occurs by direct transfer of the peptidyl chain from the tRNA bearing it to the NH2 group of the new amino acid  Translocation of the one-residue-longer peptidyl tRNA to the P site to make room for the next incoming aa- tRNA at the A site.

21. EF-Tu-EF-Ts cycle  The elongation factors are vital to cell function, so they are present in significant quantities (EF-Tu is 5% of total protein in E. coli (Table 30.8)  EF-Tu binds aminoacyl-tRNA and GTP  Aminoacyl-tRNA binds to A site of ribosome as a complex with EF-Tu and GTP  GTP is then hydrolyzed and EF-Tu:GDP complex dissociates  EF-Ts recycles EF-Tu by exchanging GTP for GDP  The elongation factors are vital to cell function, so they are present in significant quantities (EF-Tu is 5% of total protein in E. coli (Table 30.8)  EF-Tu binds aminoacyl-tRNA and GTP  Aminoacyl-tRNA binds to A site of ribosome as a complex with EF-Tu and GTP  GTP is then hydrolyzed and EF-Tu:GDP complex dissociates  EF-Ts recycles EF-Tu by exchanging GTP for GDP

24. Peptidyl Transferase  This is the central reaction of protein synthesis  23S rRNA is the peptidyl transferase!  The "reaction center" of 23S rRNA is shown in Figure 30.22 - these bases are among the most highly conserved in all of biology.  Translocation of peptidyl-tRNA from the A site to the P site follows (see Figures 30.19 & 30.21 ) catalyzed by EF-G.  This is the central reaction of protein synthesis  23S rRNA is the peptidyl transferase!  The "reaction center" of 23S rRNA is shown in Figure 30.22 - these bases are among the most highly conserved in all of biology.  Translocation of peptidyl-tRNA from the A site to the P site follows (see Figures 30.19 & 30.21 ) catalyzed by EF-G.

27. Peptide Chain Termination  Proteins known as "release factors" recognize the stop codon at the A site  Presence of release factors with a nonsense codon at A site transforms the peptidyl transferase into a hydrolase, which cleaves the peptidyl chain from the tRNA carrier  Proteins known as "release factors" recognize the stop codon at the A site  Presence of release factors with a nonsense codon at A site transforms the peptidyl transferase into a hydrolase, which cleaves the peptidyl chain from the tRNA carrier

29. The Role of GTP Hydrolysis  IF-2, EF-Tu, EF-G, RF-3 are all GTP- binding proteins  Part of the G protein superfamily  Hydrolysis drives essential conformation changes  IF-2, EF-Tu, EF-G, RF-3 interact with the same site on the 50S subunit, the factor binding center  IF-2, EF-Tu, EF-G, RF-3 are all GTP- binding proteins  Part of the G protein superfamily  Hydrolysis drives essential conformation changes  IF-2, EF-Tu, EF-G, RF-3 interact with the same site on the 50S subunit, the factor binding center

31. Polysomes  mRNA with several ribosomes  Polyribosomes  All protein synthesis occurs on polysomes  Procaryotes have around 10, eucaryotes fewer than 10 ribosomes  mRNA with several ribosomes  Polyribosomes  All protein synthesis occurs on polysomes  Procaryotes have around 10, eucaryotes fewer than 10 ribosomes