Initiation of protein synthesis is believed to be a rate-limiting step, because it requires multiple initiation factors and specific signals on messenger RNA that coordinately interact and function to form initiation complexes. The obvious difference between prokaryotic and eukaryotic mechanisms is based on the difference of where the ribosome enters the mRNA. Prokaryotic mRNAs are often polycistronic, and ribosomes can gain entry to the mRNA directly at the translation start site within the mRNA sequence to form initiation complexes, while eukaryotic mRNAs are monocistronic and ribosomes must gain entry to mRNA at its Cap and move downstream to the translation start site to form initiation complexes (see Scanning Hypothesis). Correct recognition of the site for initiation complex formation by prokaryotic ribosomes is mediated by complementary base-pairing between the Shine-Dalgarno sequence on mRNA and the anti-Shine-Dalgarno sequence on the 3′ terminus of 16S rRNA. The eukaryotic initiation process is in sharp contrast to the prokaryotic process, because it involves numerous initiation factors. This reflects the fact that specific protein factors are required for 5′ Cap recognition by the ribosome and for scanning of the ribosome to the downstream initiation site (1).
An initiation complex is first formed as a preinitiation complex composed of the small ribosomal particle (30S for prokaryotes, 40S for eukaryotes), fMet-transfer RNA, initiation factors, and GTP at the start site of mRNA. Upon formation of proper preinitiation complexes, the large ribosomal particle (50S for prokaryotes or 60S for eukaryotes) joins the preinitiation complex, to form the initiation complex (2). During this step, GTP is hydrolyzed to GDP while the fMet-tRNA-carrier factors, IF-2 (prokaryotes) and eIF-2 (eukaryotes), dissociate from the ribosome, leaving fMet-tRNA at the P site of the ribosome.
Although the translation of virtually all eukaryotic cellular mRNAs and the majority of viral RNAs is initiated via the scanning ribosome mechanism, there is a small, yet not insignificant, number of RNAs (particularly viral RNAs) that are translated by an internal initiation mechanism [see Internal Ribosome Entry Site (IRES)(3)].
Initiation of protein synthesis is a good target for regulating gene expression. Most of the bacterial ribosomal proteins are negatively autoregulated at the initiation step of their synthesis. When present in excess over rRNA, a set of ribosomal proteins (translational repressors) bind to their own mRNA and stop its translation. The ribosomal protein-binding site on its mRNA (or operator) overlaps the ribosome binding site, thereby blocking the formation of initiation complexes. The operator consists of a specific RNA structure that mimics the rRNA structure where the ribosomal protein (repressor) binds. Like the ribosomal protein repressor, E. coli threonyl-tRNA synthetase (ThrRS) is negatively autoregulated at the translation level when in excess of its substrate tRNAthr. ThrRS binds to the leader region of its own mRNA at the translational initiation site and represses its translation by preventing ribosome binding. The ThrRS operator mimics the structure tRNAthr.
Initiation of protein synthesis in eukaryotes can be regulated by phosphorylation of eIF-2a by mammalian protein kinase PKR (4). The PKR activity was originally detected as the enzyme responsible for double-stranded (ds) RNA-dependent protein synthesis inhibition in poliovirus-infected HeLa Cells, and in cell-free translation systems made from rabbit reticulocytes. The target of dsRNA’s mysterious ability to inhibit protein synthesis initiation was eventually shown to be the protein kinase PKR, which blocks protein synthesis by phosphorylating initiation factor eIF-2a. Higher eukaryotic cells possess an intrinsic defense system that is mediated by interferon. Interferon release by virus-infected cells sensitizes neighboring cells by inducing the transcription of genes that encode anti-viral products. Some of these, notably the protein kinase PKR and 2,5A synthase, act at the level of protein synthesis. (2,5A synthase makes an oligonucleotide that activates a latent ribonuclease.) Viruses, in turn, have elaborated products (e.g., the VA RNA produced by adenovirus) that neutralize these cellular defenses, thereby enabling the production of viral proteins and progeny virions.
Recruitment of the 40S small ribosomal subunit to eukaryotic mRNA is mediated by interactions between a limited set of translation initiation factors (2). One of these factors, eIF-3, is a 40S subunit-associated factor comprised of at least eight subunits in mammalian cells that interacts with the mRNA-associated initiation factor eIF-4F (Fig. 1). eIF-4F consists of two core subunits. These are the mRNA 5′-Cap-binding protein eIF-4E and the large subunit eIF-4G. Recent studies on eIF-4G have revealed that it binds to eIF-4E and eIF-3, as well as to the poly(A)-binding protein (Pab1p in yeast) (5). The multipurpose adapter nature of eIF-4G allows it to recruit the 40S ribosome to mRNA via the simultaneous association of eIF-4G with both eIF-4E and eIF-3 (Fig. 1). Knowledge of eIF-4E’s interaction with the 5′-Cap permits an understanding of cellular and viral strategies to control Cap-stimulated translation. For example, cells express a small family of inhibitory proteins that regulate eIF-4E assembly. These are called the 4E-binding proteins [4E-BPs (6)]. The 4E-BPs share an amino acid motif with the ^-terminal domain of eIF-4G that is known to be required for eIF-4G’s interaction with eIF-4E. In their nonphosphorylated form, the 4E-BPs act as competitive inhibitors of the eIF-4G-eIF-5E interaction (Fig. 1).
Figure 1. Control of initiation complex formation via Cap binding in eukaryotes (7). (a) eIF-4E recruits the 40S subunit of mRNA by protein interaction with eIF-4G and eIF-3. (b) Regulation of initiation complex formation by phosphorylati eIF-4E binding proteins, 4E-BPs.
