Prokaryote, protein synthesis and

The basic plan of protein synthesis in eukaryotes and archaea is similar to that in bacteria. The major structural and mechanistic themes recur in all domains of life. However, eukaryotic protein synthesis entails more protein components than does prokaryotic protein synthesis, and some steps are more intricate. Some noteworthy similarities and differences are as follows ... 

Despite evident similarities in mRNA translation, the molecular mechanisms underlying ribosomal recruitment and start site selection differ substantially between bacteria and eukaryotes. In most prokaryotes protein synthesis and mRNA synthesis occur simultaneously, whereas eukaryotes have decoupled gene expression by localizing transcription... 

The protein synthesis machinery reads the RNA template starting from the 5 end (the end made first) and makes proteins beginning with the amino terminus. These directionalities are set up so that in prokaryotes, protein synthesis can begin even before the RNA synthesis is complete. Simultaneous transcription-translation can t happen in eukaryotic cells because the nuclear membrane separates the ribosome from the nucleus. 

Chloramphenicol acetyl transferase (CAT). This bacterial enzyme was the first reporter protein used for studying the transcriptional activity of eukaryotic regulatory sequences (Gorman et al., 1982). CAT inactivates chloramphenicol, an inhibitor of prokaryotic protein synthesis, by converting it to the mono- or di-acetylated species. Measurement of CAT activity requires a 14C-radiolabeled chloramphenicol or acetyl-CoA and, therefore, an additional step is neccessary to separate the radio-labeled reactant from the product. Novel detection methods based on fluorescent substrates or ELISA assays, which do not use radiolabeled reagents, have been described more recently (Bullock and Gorman, 2000). 

The broad outlines of eukaryotic protein synthesis are the same as in prokaryotic protein synthesis. The genetic code is generally the same (some microorganisms and eukaryotic mitochondria use slightly different codons), rRNA and protein sequences are recognizably similar, and the same set of amino acids is used in all organisms. However, specific differences exist between the two types of protein synthesis at all steps of the process. 

Synthesis of molecular chaperones may be constitutive or stress-induced. Several size classes of molecular chaperones are synthesized constitutively to facilitate the housekeeping functions associated with protein synthesis and maturation. All organisms contain constitutively expressed chaperones, and the ubiquitous occurrence of these proteins is strong reason to believe that they appeared very early in evolution. Orthologs of some classes of molecular chaperones are found in prokaryotes and all eukaryotes. 

Figure 12.2 Initiation reactions and elongation cycle in prokaryotic protein synthesis.

Some epigenetic processes (for example protein synthesis and ribosome self-assembly) also take place in prokaryotes, and yet these cells do not give rise to embryos. It is not epigenesis as such, therefore, that accounts for development, but a particular type of epigenesis that prokaryotes do not have. It could be pointed out that prokaryotes lack the complex structures of the eukaryotic cell, but this does not explain their lack of embryonic potential. Protozoa, for example, do have the eukaryotic cell structure but they too are incapable of producing embryos. 

There are also inhibitors that affect enzyme synthesis. Inhibitors of transcription (e.g., dibromothymoquinone [DBMIB]) and inhibitors of translation (e.g., cyclo-heximide [CHX]) are available but these are not specific to particular enzymes. In addition, because protein synthesis takes place within the chloroplast and mitochondrion in eukaryotes, prokaryotic protein synthesis inhibitors (e.g., chloramphenicol [CAP]) may be necessary to distinguish prokaryotic versus eukaryotic activity (e.g., Segovia and Berges, 2005). 

The translocation step is probably the point at which prokaryotic and eukaryotic secretion differ most. The energy for this process may derive from different sources from the energy of protein synthesis in eukaryotes, and from protein synthesis and/or ATP hydrolysis and/or the membrane potential in prokaryotes. [In fact there is evidence for more than one secretion pathway in E. coli. The degree of coupling between translation and translocation may also be different in prokaryotes and eukaryotes (Section V,C).]... 

The proteins involved in the initiation of prokaryotic protein synthesis are IF-1 (binds to the A site of the 30S subunit, blocking it during initiation), IF-2 (binds to the 30S subunit and promotes the binding of the initiating tRNA to the initiation codon of mRNA), and IF-3 (prevents the 30S subunit from binding prematurely to the 50S subunit). 

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. 

Many antibiotics that are used to combat bacterial infections in humans take advantage of the differences between the mechanisms for protein synthesis in prokaryotes and eukaryotes. For example, streptomycin binds to the 30S ribosomal subunit of prokaryotes. It interferes with initiation of protein synthesis and causes misreading of mRNA. 

Many of the differences between translation in prokaryotes and eukaryotes can be seen in the response to inhibitors of protein synthesis and to toxins. The antibiotic chloramphenicol (a trade name is Ghloromycetin) binds to the A site and inhibits peptidyl transferase activity in prokaryotes, but not in eukaryotes. This property has made chloramphenicol useful in treating bacterial infections. In eukaryotes, diphtheria toxin is a protein that interferes with protein synthesis by decreasing the activity of the eukaryotic elongation factor eEF2. 

Reflect and i ply In prokaryotic protein synthesis, formylmethio-nine (fmet) is the first amino acid incorporated, whereas (normal) methionine is incorporated in eukaryotes. The same codon (AUG) serves both. What prevents methionine from being inserted into the beginning and formylmethionine in the interior ... 

Reflect and Apply What is the energy cost per amino acid in prokaryotic protein synthesis Relate this to low entropy. 

Add IPTG to E. coli cells growing in a medium containing a carbon source other than lactose in both the presence and the absence of an inhibitor of prokaryotic protein synthesis, like chloramphenicol. If zymogen activation is involved, chloramphenicol will not inhibit induction. If the synthesis of new protein is involved (as it is), induction will not be observed in the presence of chloramphenicol. 

RS. Patel et al., Badllaene, a novel inhibitor of prokaryotic protein-synthesis produced by Bacillus subtilis - Production, taxonomy, isolation, physicochemical characterisation and biological activity. J. Antibiot. 48, 997-1003 (1995)... 

RNA Ribonucleic acid linear copolymers usually of four ribonucleotides. Three major types of RNA are synthesized in the cell ribosomal RNA (rRNA), the major component of ribosomes transfer RNA (tRNA), the adaptor for protein synthesis and messenger RNA (mRNA), which is required for information transfer. Other small RNAs with specialized functions are also synthesized in small amounts in both prokaryotic and eukaryotic cells. 

FIGURE 9 Termination of protein synthesis and ribosome recycling. In prokaryotes, RF1 hydrolyzes the newly synthesized protein at stop codons UAG and UAA, while RF2 recognizes stop codons UGA and UAA. The GTPase RFS stimulates release of either RF1 or RF2. In eukaryotes a single protein recognizes all stop codons. The final step of translation is dissociation of the inactive 70S complex, stimulated by the ribosome recycling factor (RRF). 

In addition to the different translational factors and mechanisms used by eukaryotic organisms as described earlier, an organizational difference exists in eukaryotes that contrasts with prokaryotic protein synthesis. In organisms lacking a nucleus, transcription of the genetic message from DNA to RNA occurs in the same location as translation. In fact, bacterial ribosomes typically be-... 

Chloramphenicol (Cm) is a broad spectrum antibiotic that acts by inhibiting the function of bacterial ribosomes. Cm binds the 50 subunit of bacterial (70S) ribosomes and inhibits peptidyltransferase (Kj 3 ixM), effectively blocking prokaryotic protein synthesis. Because of its broad spectrum activity on ribosomal function, Cm also causes some serious side effects in eukaryotic hosts. (In humans... 

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