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

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 Met-tRNAm

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)

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

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 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

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.

EF-Tu-EF-Ts cycleEF-Tu-EF-Ts cycle

The The elongation factorselongation factors are vital to cell are vital to cell function, so they are present in significant function, so they are present in significant quantities (EF-Tu is 5% of total protein in quantities (EF-Tu is 5% of total protein in E. E. colicoli (Table 30.8) (Table 30.8)

EF-Tu binds aminoacyl-tRNA and GTP EF-Tu binds aminoacyl-tRNA and GTP Aminoacyl-tRNA binds to A site of ribosome Aminoacyl-tRNA binds to A site of ribosome

as a complex with EF-Tu and GTP as a complex with EF-Tu and GTP GTP is then hydrolyzed and EF-Tu:GDP GTP is then hydrolyzed and EF-Tu:GDP

complex dissociatescomplex dissociates EF-Ts recycles EF-Tu by exchanging GTP for EF-Ts recycles EF-Tu by exchanging GTP for

GDPGDP

The The elongation factorselongation factors are vital to cell are vital to cell function, so they are present in significant function, so they are present in significant quantities (EF-Tu is 5% of total protein in quantities (EF-Tu is 5% of total protein in E. E. colicoli (Table 30.8) (Table 30.8)

EF-Tu binds aminoacyl-tRNA and GTP EF-Tu binds aminoacyl-tRNA and GTP Aminoacyl-tRNA binds to A site of ribosome Aminoacyl-tRNA binds to A site of ribosome

as a complex with EF-Tu and GTP as a complex with EF-Tu and GTP GTP is then hydrolyzed and EF-Tu:GDP GTP is then hydrolyzed and EF-Tu:GDP

complex dissociatescomplex dissociates EF-Ts recycles EF-Tu by exchanging GTP for EF-Ts recycles EF-Tu by exchanging GTP for

GDPGDP

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 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

PolysomesPolysomes

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