Protein synthesis prokaryotic initiation factors

Even though specific differences distinguish the initiation process in eukaryotes and prokaryotes, three things must be accomplished to initiate protein synthesis in all systems (1) The small ribosomal subunit must bind the initiator tRNA (2) the appropriate initiating codon on mRNA must be located and (3) the large ribosomal subunit must associate with the complex of the small subunit, the initiating tRNA, and mRNA. Nonribosomal proteins, known as initiation factors (IFs), participate in each of these three processes. IFs interact transiently with a ribosome during initiation and thus differ from ribosomal proteins, which remain continuously associated with the same ribosome. 

Prokaryotic initiation factors. In addition to the ribosomal proteins, the initiation factors IFl, IF2, and IF3, whose molecular masses are 9.5, 9.7, and 19.7 kDa, respectively, " are essential. They coordinate a sequence of reactions that begins with the dissociation of 70S ribosomes into their 30S and 50S subunits. Then, as is shown in Fig. 29-10, the mRNA, the initiator tRNA charged with formylmethionine, the three initiation factors, and the ribosomal subunits react to form 70S programmed ribosomes, which carry the bound mRNA and are ready to initiate protein synthesis. IF2 is a specialized G protein (Chapter 11), which binds and hydrolyzes GTP. It resembles the better known elongation factor EF-Tu (Section 2). The 172-residue IF3 consists of two compact a/p domains linked by a flexible sequence, which may exist as an a Its C-terminal domain binds to the central domain of the 16S RNA near nucleotides 819-859 (Fig. 

Initiation of protein synthesis involves the assembly of the components of the translation system before peptide bond formation occurs. These components include the two ribosomal subunits, the mRNA to be translated, the aminoacyl-tRNA specified by the first codon in the message, GTP (which provides energy for the process), and initiation factors that facilitate the assembly of this initiation complex (see Figure 31.13). [Note In prokaryotes, three initiation factors are known (IF-1, IF-2, and IF-3), whereas in eukary- otes, there are at least ten (designated elF to indicate eukaryotic origin).] There are two mechanisms by which the ribosome recognizes the nucleotide sequence that initiates translation ... 

Formation of the initiation complex for protein synthesis in prokaryotes. E. coli has three initiation factors bound to a pool of 30S ribosomal subunits. One of these factors, IF-3, holds the 30S and 50S subunits apart after termination of a previous round of protein synthesis. The other two factors, IF-1 and IF-2, promote the binding of both fMet-tRNAfMel and mRNA to the 30S subunit. The binding of mRNA occurs so that its Shine-Dalgamo sequence pairs with 16S... 

Formation of the initiation complex for protein synthesis in eukaryotes. The reaction begins with the small subunit held apart from the large subunit by an antiassociation factor and ends with the hydrolysis of GTP and joining of the large subunit as in prokaryotes. The intervening reactions are different. A much more complex spectrum of initiation factors (elFs) is involved, and the exact function of only a few of these factors is known with certainty. The... 

At the conclusion of the initiation process, the ribosome is poised to translate the reading frame associated with the initiator codon. The translation of the contiguous codons in mRNA is accomplished by the sequential repetition of three reactions with each amino acid. These three reactions of elongation are similar in both prokaryotic and eukaryotic systems two of them require nonribosomal proteins known as elongation factors (EF). Interestingly, the actual formation of the peptide bond does not require a factor and is the only reaction of protein synthesis catalyzed by the ribosome itself. 

Initiation of protein synthesis is catalyzed by proteins called initiation factors (IFs). In prokaryotes, three initiation factors (IF1, IF2 and IF3) are essential. Because of the complexity of the process, the exact order of binding of IF1, IF2, IF3, fMet-tRNAfMet and mRNA is still unclear. One current model is shown in Fig. 4 and is described below. 

Initiation of protein synthesis in eukaryotes requires at least nine distinct eukaryotic initiation factors (elFs) (see Table 1) compared to the three initiation factors (IFs) in prokaryotes (see Topic H2). 

Both prokaryotes and eukaryotes initiate protein synthesis with a specialized methionyl-tRNA in response to an AUG initiation codon. Eukaryotes, however, use an initiator met—tRNAmeti—that is not formylated. Recognition of the initiator AUG is also different. Only one coding sequence exists per eukaryotic mRNA, and eukaryotic mRNAs are capped. Initiation, therefore, uses a specialized capbinding initiation factor to position the mRNA on the small riboso-mal subunit. Usually, the first AUG after the cap (that is, 3 to it) is used for initiation. 

Protein synthesis takes place in three phases initiation, elongation, and termination. In prokaryotes, mRNA, formylmethionyl-tRNAf (the special initiator tRNA that recognizes AUG), and a 308 ribosomal suhunit come together with the assistance of initiation factors to form a 308 initiation complex. A 508 rihosomal suhunit then joins this complex to form a 708 initiation complex, in which fMet-tRNAf occupies the P site of the rihosome. 

Figure 3-25. The initiation (A) and elongation (B) reactions of protein synthesis. EF-1 and EF-2 are eukaryotic elongation factors corresponding to EF-Tu and EF-G in prokaryotes.

C. mRNA supplies the codons, aminoacyl-tRNA and GTP provide energy, peptidyl transferase catalyzes the formation of peptide bonds, and elongation factor 2 translocates the peptidyl-tRNA. Formylmethionyl-tRNA is involved in initiation of protein synthesis in prokaryotes. 

Figure 12.4 outlines the process of protein synthesis involving the ribosome, mRNA, a series of amino-acyl transfer RNA (tRNA) molecules (at least one for each amino acid) and accessory protein factors involved in initiation, elongation and termination. As the process is essentially the same in prokaryotic (bacterial) and eukaryotic cells (i.e. higher organisms and mammalian cells) it is surprising that there are so many selective agents which act in this area (see Fig. 12.1). 

A list of key differences between prokaryotes and eukaryotes with respect to protein synthesis is shown in Table 9-1. These include the existence of multiple eukaryotic initiation factors that facilitate the assembly of the riboso-mal protein synthetic machinery, whereas there are only three for prokaryotes. An initiation site on bacterial mRNA consists of the AUG initiation codon preceded with a gap of approximately 10 bases by the Shine-Dalgamo polypurine hexamer, whereas the 5 Cap (a 7-methylguanylate residue in a 5 —>5 triphosphate linkage) acts as an initiation signal in eukaryotes. In prokaryotes, the first or A-terminal amino acid is a formyl-methionine (fMet), but in eukaryotes it is usually a simple methionine. Additionally, the size and nature of the prokaryotic ribosomes are quite different from the eukaryotic ribosomes. 

Eukaryotic protein synthesis is slower and more complex than its prokaryotic counterpart. In addition to requiring a larger number of translation factors and a more complex initiation mechanism, the eukaryotic process also involves vastly more complicated posttranslational processing and targeting mechanisms. Eukaryotes use a wide spectrum of translational control mechanisms. 

In this section, we describe the three basic stages of protein synthesis initiation, elongation, and termination. These three processes are fairly similar between prokaryotes and eukaryotes, with the two exceptions being that more protein factors have been identified as necessary for eukaryotic protein synthesis, and that transcription and translation are physically linked in prokaryotes but not in eukaryotes. Note that the reactions will be schematized as a single ribosome transversing the mRNA, but as shown in Figure 26.3, translation actually occurs on polyribosomes. 

Plants, in common with microorganisms and animals, require methionine chiefly for three roles, (a) As a component of protein, a role which accounts for most of the methionine in the cell, (b) As a component of methionyl tRNA (in eukaryotes) and formylmethionyl tRNA (in chloroplasts, mitochondria, and prokaryotes), factors required for initiation of protein synthesis. (c) As a component of AdoMet, the chief biological methyl donor, the obligatory precursor of spermidine and spermine, and an effector of certain enzymes. In addition to these chief roles, a major pathway for the metabolism of methionine in certain plant tissues is its conversion to ethylene (see Yang and Adams, this series, Vol. 4, Chapter 6). Only plants and microorganisms can synthesize the homocysteine moiety of methionine novo, and the importance of this synthesis in the sulfur cycle has been noted in the introduction. 

Fig. 2. Formation of a stable initiation complex between a 70 S ribosome and messenger RNA. In the final complex fMet-tRNAf " is in the correct position for the formation of a peptide bond. IF-1, IF-2, and IF-3 are the protein initiation factors and fMet-tRNAf " is the formyl methionyl tRNA which is used for the initiation of protein synthesis in prokaryotes. The process in animal cells is thought to be substantially the same, the initiation factors being termed IF-Ml, IF-M2, and IF-M3 and the initiator tRNA, Met-tRNAt . The methionine attached to this tRNA species is not normally formylated but can be so modified by enzymes from bacterial cells.

Assembly of the ribosomal subunits, mRNA, and initiator tRNA into a complex ready for protein synthesis requires several proteins called initiation factors. In prokaryotes, three initiation factors (IFs) transiently associate with the components of the translational machinery IFl, IF2, and IF3. (In eukaryotes, more factors are required but the overall initiation process is similar with a few exceptions described below.) Table II summarizes the properties of E. coli initiation factors as well as protein factors involved in elongation and termination. 

The basal transcription complex described in Chapter 29 initiates transcription at a low frequency. Recall that several general transcription factors (the preinitiation complex) join with KNA polymerase II to form the transcription complex. Additional transcription factors must bind to other sites for a gene to achieve a high rate of mRNA synthesis. In contrast with the regulators of prokaryotic transcription, few eukaryotic transcription factors have any effect on transcription on their own. Instead, each factor recruits other proteins to build up large complexes that interact with the... 

The answer is e. (Murray, pp 435-451. Scriver, pp 3-45. Sack, pp 1-40. Wilson, pp 101—120.) The first event that occurs in mRNA synthesis is the binding of transcription factor TFllD to the TATA box. This consensus sequence portion of virtually all eukaryotic genes coding for mRNA is centered at about —25 and is similar to a 10-sequence promoter box found in prokaryotes. TFllD contains a TATA box-binding protein. The following sequence occurs in the initiation of mRNA synthesis ... 

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