Figure 27.13 shows the components of the E. coli ribosome (Figure AO1). Eukaryotic ribosomes are similar, but somewhat more complex in structure: The 80S ribosome has 60S and 40S subunits. The 60S subunit has three rRNAs (28S [4800 nucleotides], 5.8S [160 nucleotides], and 5S [120 nucleotides]) and 50 proteins. The 40S subunit has an 18S [1900 nucleotides] rRNA and 33 proteins. For paper on structure of the ribosome (html version) (pdf version).
Figure AO shows the structure of the initiator fmet-tRNAf. Methionine is attached to both tRNAf and tRNAm by the same aminoacyl-tRNA synthetase. Met-tRNAf is further converted by a specific enzyme to fmet-tRNAf. Met-tRNAm is not a substrate for this extra reaction. fmet-tRNAf is recognized by IF-2 and is the first amino acid at the amino-terminus of the polypeptide chain. Met-tRNAm is recognized by EF-Tu and is placed at internal positions of the polypeptide chain. Both tRNAs interact with the codon AUG (although tRNAf can also use UUG and GUG during initiation).
Table 27.3 shows Shine-Dalgarno sequences: interactions between 16S rRNA sequences and mRNA sequences. Messages with mutated Shine-Dalgarno sequences are poorly translated.
Mutations in the Shine-Dalgarno sequence:
The CCUCC sequence in one of the 16S rRNA genes was changed drastically to GGAGG or CACAC, and used in vivo to translate human growth hormone (hGH) mRNA containing a CCUCC or GUGUG sequence upstream from its start codon. Translation of the hGH mRNA depended on complementarity between the corresponding 16S rRNA and mRNA sequence. Since normal mRNAs were not translated by the "genetically engineered ribosomes," specific proteins can therefore be targeted to be overproduced.
Initiation factors, elongation factors, and release factors are members of the superfamiliy of proteins called G proteins. G proteins are GTPases and regulate a wide variety of cellular processes according to a basic reaction cycle. They adopt an "on" conformation when bound to GTP. In this state they can bind to another macromolecule or complex and trigger a distict reaction. After a G protein has done its job, the intrinsic GTPase hydrolyzes GTP to GDP and Pi , and the resulting GDP induces an "off" conformation that leads to the dissociation of the G protein from the macromolecule or complex. In the case of G protein factors involved in translation, the GTPase activity is ribosome-dependent (and probably catalyzed by ribosomal components rather than the factors themselves).
(See Table 27.4 on page 1044) The textbook has the roles of IF-1 and IF-3 reversed!
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| Factor | Binds GTP |
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| IF-1 | No |
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| IF-2 | Yes |
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| IF-3 | No |
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Initiation in bacteria involves the interaction of the 30S subunit with the Shine-Dalgarno sequence on mRNA that is complementary to the 3' end of 16S RNA. The process also involves three initiation factors, IF1, IF2, and IF3. IF3 is known to bind strongly to the 30S subunit and prevent its association with the 50S subunit. It also helps in the selection of initiator tRNA (fMet-tRNAf by destabilizing the binding of other tRNAs in the P site of the ribosome. In a possibly related function, IF3 has been found to dissociate deacylated tRNA from the 30S subunit in the last step of termination before it is recycled in a new round of protein synthesis. IF2 is a GTPase that binds preferentially to fmet-tRNAf, and its affinity for the ribosome is increased by IF1. Surprisingly, recent kinetic data indicate that the GTPase activity of IF2 is required neither for the proper placement of initiator tRNA in the P site nor for IF2 release. Structures of bacterial IF1, IF3, and an archaebacterial IF2 homolog (eIF-5B) have been solved.
The crystal structure of the 30S-IF1 complex
shows that IF1 binds to the A site of the 30S ribosomal subunit,
consistent with previous biochemical data. In doing so, it prevents
tRNA binding in the A site, but also induces a conformational
change that may represent the transition state in the equilibrium
between subunit association and dissociation. The location of
IF3 is still controversial. No direct location of IF2 has been
determined, but since it is known to bind the aminoacyl end of
initiator tRNA in the P site, as well as interact with IF1, a
model could be proposed in which it binds over IF1 in the A site.
In addition, presumably its GTPase domain binds in the vicinity
of the factor binding site of the 50S subunit where the corresponding
domains of elongation factors G and Tu (EF-G and EF-Tu) also bind,
since it is known to footprint some of the same residues in 23S
RNA. When one combines the current structural and biochemical
data, a view emerges in which IF1 binds in the A site, IF2 binds
over the A site, the P site is occupied by initiator tRNA, and
IF3 occupies the E site. Thus, all of the tRNA sites are occupied
in the initiation complex, presumably "setting" the
correct conformation of the 30S for the initiation of protein
synthesis. However, this raises a number of questions. Why do
all of the tRNA binding sites need to be occupied? If the GTPase
activity of IF2 is not required for P-site tRNA binding or for
IF2 release, what is its role? When does the 50S subunit become
associated with the initiation complex? Finally, despite many
years of work, the order in which the factors bind and are released
in vivo, and what they have to do with the conformation of the
ribosome, have not been definitively elucidated.
Figure 27.20 gives an overview of the process of initiation of translation (but remember that IF-1 and IF-3 should be exchanged on this diagram!).
Initiation almost always uses the first AUG downstream from the 5'-cap as the start codon, but in the context of the sequence GCC(A or G)CCAUGG. The most important features of this sequence are the purine (A or G) 3 bases before the AUG and the G immediately following it (together they influence the efficiency of translation by 10-fold). The initiator met-tRNAi differs structurally from the met-tRNAm used in elongating the protein (but methionine is NOT formylated as in E. coli!).
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GTPase activity stimulated by eIF-5 |
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eIF-4F complex:
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eIF-4F
complex:
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eIF-4F
complex:
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and the release of eIF-2-GDP |
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The structure m7GpppN (or the cap, where m is a methyl group and N any nucleotide) is present at the 5' end of all nuclear transcribed mRNAs, and plays an important role in the initiation process. The cap is recognized by the initiation factor eIF4E (Figure BC). eIF4E, via an interaction with a large scaffolding protein termed eIF4G, directs the translational machinery to the 5' end of the mRNA. eIF4E and eIF4G function as components of a trimeric complex, termed eIF4F, which also contains the RNA helicase eIF4A. eIF4G also establishes intermolecular contacts with several other components of the translational machinery: for example, the poly A binding protein and the multisubunit, ribosome-associated initiation factor eIF3. Importantly, optimal binding of the 40S ribosomal subunit is thought to require a region of single-stranded mRNA. Thus, once eIF4F binds to the cap, eIF4A (in conjunction with an associated cofactor, eIF4B) is thought to unwind any inhibitory secondary structure present in the cap-proximal 5' untranslated region (5'UTR). Through its interaction with eIF3 and its ability to bind mRNA in a sequence nonspecific fashion, eIF4G bridges the mRNA to the 40S ribosomal subunit. Once the 40S subunit is bound to mRNA, the ribosomal subunit and other associated factors (eIF-1A, eIF-2-GTP-met-tRNAi) are believed to scan in a 5' to 3' direction, until an initiation codon in the proper sequence context is encountered. When an initiation codon is recognized, and a codon/anticodon interaction established with met-tRNAi, GTP hydrolysis to GDP and Pi by eIF-2 (assisted by eIF-5) and eIF-5B allows the initiation factors to dissociate from the small ribosomal subunit and subsequent joining to a 60S ribosomal subunit. The requirement in eukaryotes for both eIF2 and eIF-5B suggests that two molecules of GTP are hydrolyzed during each round of translation initiation, which provides and additional entry point for regulation of eukaryotic gene expression that is not present in bacteria. Figure 28.35 shows the overall process of eukaryotic initiation of protein synthesis (this figure has been extensively up-dated from the textbook version)!