- A ribosome-binding site is a sequence on bacterial mRNA that includes an initiation codon that is bound by a 30S subunit in the initiation phase of protein synthesis.
- An initiation complex in bacterial protein synthesis contains a small ribosome subunit, initiation factors, and initiator aminoacyl-tRNA bound to mRNA at an AUG initiation codon.
- Initiation factors (IF) (IF in prokaryotes, eIF in eukaryotes) are proteins that associate with the small subunit of the ribosome specifically at the stage of initiation of protein synthesis.
- IF-1 is a bacterial initiation factor that stabilizes the initiation complex.
- IF-2 is a bacterial initiation factor that binds the initiator tRNA to the initiation complex.
- IF-3 is a bacterial initiation factor required for 30S subunits to bind to initiation sites in mRNA. It also prevents 30S subunits from binding to 50S subunits.
- Initiation of protein synthesis requires separate 30S and 50S ribosome subunits.
- Initiation factors (IF-1,2,3), which bind to 30S subunits, are also required.
- A 30S subunit carrying initiation factors binds to an initiation site on mRNA to form an initiation complex.
- IF-3 must be released to allow 50S subunits to join the 30S-mRNA complex.
Bacterial ribosomes engaged in elongating a polypeptide chain exist as 70S particles. At termination, they are released from the mRNA as free ribosomes. In growing bacteria, the majority of ribosomes are synthesizing proteins; the free pool is likely to contain ~20% of the ribosomes.
Ribosomes in the free pool can dissociate into separate subunits; so 70S ribosomes are in dynamic equilibrium with 30S and 50S subunits. Initiation of protein synthesis is not a function of intact ribosomes, but is undertaken by the separate subunits, which reassociate during the initiation reaction. Figure 6.9 summarizes the ribosomal subunit cycle during protein synthesis in bacteria.
Initiation occurs at a special sequence on mRNA called the ribosome-binding site. This is a short sequence of bases that precedes the coding region (see Figure 6.16). The small and large subunits associate at the ribosome-binding site to form an intact ribosome. The reaction occurs in two steps:
- Recognition of mRNA occurs when a small subunit binds to form an initiation complex at the ribosome-binding site.
- Then a large subunit joins the complex to generate a complete ribosome.
Although the 30S subunit is involved in initiation, it is not by itself competent to undertake the reactions of binding mRNA and tRNA. It requires additional proteins called initiation factors (IF). These factors are found only on 30S subunits, and they are released when the 30S subunits associate with 50S subunits to generate 70S ribosomes. This behavior distinguishes initiation factors from the structural proteins of the ribosome. The initiation factors are concerned solely with formation of the initiation complex, they are absent from 70S ribosomes, and they play no part in the stages of elongation (for review see Maitra et al., 1982). Figure 6.10 summarizes the stages of initiation.
Bacteria use three initiation factors, numbered IF-1, IF-2, and IF-3. They are needed for both mRNA and tRNA to enter the initiation complex:
- IF-3 is needed for 30S subunits to bind specifically to initiation sites in mRNA.
- IF-2 binds a special initiator tRNA and controls its entry into the ribosome.
- IF-1 binds to 30S subunits only as a part of the complete initiation complex. It binds to the A site and prevents aminoacyl-tRNA from entering (Moazed et al., 1995). Its location also may impede the 30S subunit from binding to the 50S subunit (Carter et al., 2001).
IF-3 has multiple functions: it is needed first to stabilize (free) 30S subunits; then it enables them to bind to mRNA; and as part of the 30S-mRNA complex it checks the accuracy of recognition of the first aminoacyl-tRNA (see 6.6 Use of fMet-tRNAf is controlled by IF-2 and the ribosome.
The first function of IF-3 controls the equilibrium between ribosomal states, as shown in Figure 6.11. IF-3 binds to free 30S subunits that are released from the pool of 70S ribosomes. The presence of IF-3 prevents the 30S subunit from reassociating with a 50S subunit. The reaction between IF-3 and the 30S subunit is stoichiometric: one molecule of IF-3 binds per subunit. There is a relatively small amount of IF-3, so its availability determines the number of free 30S subunits.
IF-3 binds to the surface of the 30S subunit in the vicinity of the A site. There is significant overlap between the bases in 16S rRNA protected by IF-3 and those protected by binding of the 50S subunit, suggesting that it physically prevents junction of the subunits (Dallas and Noller, 2001). IF-3 therefore behaves as an anti-association factor that causes a 30S subunit to remain in the pool of free subunits.
The second function of IF-3 controls the ability of 30S subunits to bind to mRNA. Small subunits must have IF-3 in order to form initiation complexes with mRNA. IF-3 must be released from the 30S·mRNA complex in order to enable the 50S subunit to join. On its release, IF-3 immediately recycles by finding another 30S subunit.
IF-2 has a ribosome-dependent GTPase activity: it sponsors the hydrolysis of GTP in the presence of ribosomes, releasing the energy stored in the high-energy bond. The GTP is hydrolyzed when the 50S subunit joins to generate a complete ribosome. The GTP cleavage could be involved in changing the conformation of the ribosome, so that the joined subunits are converted into an active 70S ribosome.