Small subunits scan for initiation sites on eukaryotic mRNA

KEY CONCEPTS:

• Eukaryotic 40S ribosomal subunits bind to the 5 end of mRNA and scan the mRNA until they reach an initiation site.
• A eukaryotic initiation site consists of a 10 nucleotide sequence that includes an AUG codon.
• 60S ribosomal subunits join the complex at the initiation site.

Initiation of protein synthesis in eukaryotic cytoplasm resembles the process in bacteria, but the order of events is different, and the number of accessory factors is greater. Some of the differences in initiation are related to a difference in the way that bacterial 30S and eukaryotic 40S subunits find their binding sites for initiating protein synthesis on mRNA. In eukaryotes, small subunits first recognize the 5 end of the mRNA, and then move to the initiation site, where they are joined by large subunits. (In prokaryotes, small subunits bind directly to the initiation site.)

Virtually all eukaryotic mRNAs are monocistronic, but each mRNA usually is substantially longer than necessary just to code for its protein. The average mRNA in eukaryotic cytoplasm is 1000-2000 bases long, has a methylated cap at the 5 terminus, and carries 100-200 bases of poly(A) at the 3 terminus.

The nontranslated 5 leader is relatively short, usually <100 bases. The length of the coding region is determined by the size of the protein. The nontranslated 3 trailer is often rather long, sometimes ~1000 bases.

The first feature to be recognized during translation of a eukaryotic mRNA is the methylated cap that marks the 5 end. Messengers whose caps have been removed are not translated efficiently in vitro. Binding of 40S subunits to mRNA requires several initiation factors, including proteins that recognize the structure of the cap.

Modification at the 5 end occurs to almost all cellular or viral mRNAs, and is essential for their translation in eukaryotic cytoplasm (although it is not needed in organelles). The sole exception to this rule is provided by a few viral mRNAs (such as poliovirus) that are not capped; only these exceptional viral mRNAs can be translated in vitro without caps. They use an alternative pathway that bypasses the need for the cap.

Some viruses take advantage of this difference. Poliovirus infection inhibits the translation of host mRNAs. This is accomplished by interfering with the cap binding proteins that are needed for initiation of cellular mRNAs, but that are superfluous for the noncapped poliovirus mRNA.

We have dealt with the process of initiation as though the ribosome-binding site is always freely available. However, its availability may be impeded by secondary structure. The recognition of mRNA requires several additional factors; an important part of their function is to remove any secondary structure in the mRNA (see Figure 6.20).

Sometimes the AUG initiation codon lies within 40 bases of the 5 terminus of the mRNA, so that both the cap and AUG lie within the span of ribosome binding. But in many mRNAs the cap and AUG are farther apart, in extreme cases ~1000 bases distant. Yet the presence of the cap still is necessary for a stable complex to be formed at the initiation codon. How can the ribosome rely on two sites so far apart?

Figure 6.18 illustrates the "scanning" model, which supposes that the 40S subunit initially recognizes the 5 cap and then "migrates" along the mRNA. Scanning from the 5 end is a linear process. When 40S subunits scan the leader region, they can melt secondary structure hairpins with stabilities <–30 kcal, but hairpins of greater stability impede or prevent migration.

Migration stops when the 40S subunit encounters the AUG initiation codon. Usually, although not always, the first AUG triplet sequence to be encountered will be the initiation codon. However, the AUG triplet by itself is not sufficient to halt migration; it is recognized efficiently as an initiation codon only when it is in the right context. The most important determinants of context are the bases in positions –4 and +1. An initiation codon may be recognized in the sequence NNNPuNNAUGG. The purine (A or G) 3 bases before the AUG codon, and the G immediately following it, can influence the efficiency of translation by 10×. When the leader sequence is long, further 40S subunits can recognize the 5 end before the first has left the initiation site, creating a queue of subunits proceeding along the leader to the initiation site (Kozak, 1978; for review see Kozak, 1983).

It is probably true that the initiation codon is the first AUG to be encountered in the most efficiently translated mRNAs. But what happens when there is an AUG triplet in the 5 nontranslated region? There are two possible escape mechanisms for a ribosome that starts scanning at the 5 end. The most common is that scanning is leaky, that is, a ribosome may continue past a non-initiation AUG because it is not in the right context. In the rare case that it does recognize the AUG, it may initiate translation but terminate before the proper initiation codon, after which it resumes scanning.

The vast majority of eukaryotic initiation events involve scanning from the 5 cap, but there is an alternative means of initiation, used especially by certain viral RNAs, in which a 40S subunit associates directly with an internal site called an IRES (for review see Hellen and Sarnow, 2001). (This entirely bypasses any AUG codons that may be in the 5 nontranslated region.) There are few sequence homologies between known IRES elements. We can distinguish three types on the basis of their interaction with the 40S subunit:

• One type of IRES includes the AUG initiation codon at its upstream boundary. The 40S subunit binds directly to it, using a subset of the same factors that are required for initiation at 5 ends (Kaminski, Howell, and Jackson, 1990; Pestova, Hellen, and Shatsky, 1996).
• Another is located as much as 100 nucleotides upstream of the AUG, requiring a 40S subunit to migrate, again probably by a scanning mechanism.
• An exceptional type of IRES in hepatitis C virus can bind a 40S subunit directly, without requiring any initiation factors (Pestova et al., 1998). The order of events is different from all other eukaryotic initiation. Following 40S - mRNA binding, a complex containing initiator factors and the initiator tRNA binds.

Use of the IRES is especially important in picornavirus infection, where it was first discovered, because the virus inhibits host protein synthesis by destroying cap structures and inhibiting the initiation factors that bind them (see 6.9 Eukaryotes use a complex of many initiation factors)(Pelletier and Sonenberg, 1988).

Binding is stabilized at the initiation site. When the 40S subunit is joined by a 60S subunit, the intact ribosome is located at the site identified by the protection assay. A 40S subunit protects a region of up to 60 bases; when the 60S subunits join the complex, the protected region contracts to about the same length of 30-40 bases seen in prokaryotes.