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Initiation in Bacteria Needs 30S Subunits and Accessory Factors


  

2501       11:00 صباحاً       التاريخ: 23-5-2021              المصدر: JOCELYN E. KREBS, ELLIOTT S. GOLDSTEIN and STEPHEN T. KILPATRICK
Initiation in Bacteria Needs 30S Subunits and Accessory Factors

KEY CONCEPTS
- Initiation of translation in prokaryotes requires separate 30S and 50S ribosomal subunits.
- Initiation also requires initiation factors (IF-1, IF-2, and IF-3), which bind to 30S subunits.
- A 30S subunit carrying initiation factors binds to an initiation site on the mRNA to form an initiation complex.
- IF-3 must be released to allow the 50S subunit to join the 30S-mRNA complex.
Prokaryotic ribosomes engaged in elongating a polypeptide chain exist as 70S particles. At termination, they are released from the mRNA as free ribosomes or ribosomal subunits. In growing bacteria, the majority of ribosomes are synthesizing polypeptides; the free pool is likely to contain about 20% of the ribosomes.
Ribosomes in the free pool can dissociate into separate subunits; this means that 70S ribosomes are in dynamic equilibrium with 30S and 50S subunits. Initiation of translation is not a function of intact ribosomes, but is undertaken by the separate subunits. These subunits reassociate during the initiation reaction. Figure 1 summarizes the ribosomal subunit cycle during translation in bacteria.
src=../../../medea/images/2_841.jpg
FIGURE 1. Initiation requires free ribosome subunits. When ribosomes are released at termination, the 30S subunits bind initiation factors and dissociate to generate free subunits. When subunits reassociate to produce a functional ribosome at initiation, they release these factors.
Initiation occurs at a special sequence on mRNA called the ribosome-binding site (including the Shine–Dalgarno sequence, which is discussed in the next section). This is a short sequence of bases that is positioned upstream from the coding region and is complementary to a portion of the 16S rRNA (see the section later in this chapter titled 16S rRNA Plays an Active Role in Translation). 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.
- A large subunit then joins the complex to generate a complete ribosome.
Although the 30S subunit is involved in initiation, it is not sufficient by itself to bind mRNA and tRNA; this requires additional proteins called initiation factors (IFs). 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 action distinguishes initiation factors from the structural proteins of the ribosome. The initiation factors are solely concerned with formation of the initiation complex; they are absent from 70S ribosomes and they play no part in the stages of elongation. Figure 2 summarizes the stages of initiation.
src=../../../medea/images/2_842.jpg
FIGURE 2. Initiation factors stabilize free 30S subunits and bind initiator tRNA to the 30S–mRNA complex.
Prokaryotes 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 has multiple functions: It is needed to stabilize (free) 30S subunits and to inhibit the premature binding of the 50S subunit; it enables 30S subunits to bind to initiation sites in mRNA; and, as part of the 30S-mRNA complex, it checks the accuracy of recognition of the first aminoacyl-tRNA.
- IF-2 binds a special initiator tRNA and controls its entry into the ribosome.
- IF-1 binds to 30S subunits as a part of the complete initiation complex. It binds in the vicinity of the A site and prevents aminoacyl-tRNA from entering. Its location also may impede the 30S subunit from binding to the 50S subunit.
Numerous structural studies indicate that IF-3 has two distinct, largely globular domains, with the C-terminal domain at the 50S contact site on the 30S subunit and the N-terminal domain in the vicinity of the 30S E site. This broad positioning of IF-3 on the 30S subunit is consistent with its multiple functions.
The first function of IF-3 is control of the equilibrium between ribosomal states, as shown in Figure 3. 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. IF-3 can interact directly with 16S rRNA, and significant overlap exists 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. IF-3 therefore behaves as an anti-association factor that causes a 30S subunit to remain in the pool of free subunits. The reaction between IF-3 and the 30S subunit is stoichiometric: One molecule of IF-3 binds per subunit. Because of the relatively small amount of IF-3, its availability determines the number of free 30S subunits.
src=../../../medea/images/2_843.jpg
FIGURE 3. Initiation requires 30S subunits that carry IF-3.
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 for the 50S subunit to join. On its release, IF-3 immediately recycles by finding another 30S subunit. Finally, IF-3 checks the accuracy of recognition of the first aminoacyl-tRNA and helps to direct it to the P site of the 30S subunit. The former has been attributed to the C-terminal domain of IF-3 . By comparison, the Nterminal domain of IF-3 is positioned to help direct the aminoacyltRNA into the P site of the 30S subunit by blocking the E site at the same time that IF-1 is blocking the A site.

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.


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