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Protein synthesis
 30.4 Ribosome Structure and Assembly
 30.5 Mechanics of Protein Synthesis
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
Mg2+
 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
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
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
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
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
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
Prokaryotic Initiation
 The initiator tRNA is one with a formylated
methionine: f-Met-tRNAfMet
 It is only used for initiation, and regular MettRNAmMet 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)
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-MettRNAfMet, mRNA and 30S ribosome form the 30S
initiation complex
Events of Initiation
 30S subunit with IF-1 and IF-3 binds mRNA, IF-2,
GTP and f-Met-tRNAfMet (Figure 30.18)
 IF-2 delivers the initiator tRNA in a GTPdependent 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
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 aatRNA at the A site.
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
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.
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
The Role of GTP Hydrolysis
 IF-2, EF-Tu, EF-G, RF-3 are all GTPbinding 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
Polysomes
 mRNA with several ribosomes
 Polyribosomes
 All protein synthesis occurs on
polysomes
 Procaryotes have around 10,
eucaryotes fewer than 10 ribosomes