Termination in Prokaryotes

Be able to describe the mechanism for peptide chain initiation, elongation, and termination in prokaryotes and eukaryotes on the ribosome. 

Figure 23-6 Schematic diagram of translational termination in prokaryotes.

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. 

The mechanism of protein biosynthesis occurs in three consecutive phases initiation, elongation and termination. In prokaryotes and eukaryotes, differences exist in the mechanisms employed. These differences relate to ... 

Specific codons in mRNA signal the initiation and termination of synthesis of each protein. And there exist within the ribosome a number of factors which permit the stepwise synthesis of the polypeptide. In addition, soluble factors are required for initiation, elongation, translocation, and termination. In prokaryotes, translation is closely linked temporally to transcription, since both processes take place in the same milieu in eukaryotes, the nucleus compartmentalizes these two processes, permitting further controls and delaying the coupling of translation to transcription. 

PelZ is a hydrophilic protein of 420 amino acids with a short hydrophobic sequence at its N-terminal end which has Ae characteristics of the signal sequences of exported proteins. The signal peptide may be 24 amino acids long, which would corroborate wiA the usual length encountered in prokaryotes. The molecular cloning of the pelZ gene in an expression vector pT7-6 allowed for the specific 35S-cysteine-methionine raAo-labelling of PelZ in E. coli K38. We could detect, in crude extracts, the presence of a precursor and a mature form of PelZ. After cell fractionation, Ae mature form of PelZ could be localized in Ae periplasm of E. coli. So PelZ appears to be a protein exported by Ae Sec-dependent system of translocation. 

Another factor that characterizes molybdenum and tungsten enzymes is that instead of using the metal itself, directly coordinated to amino acid side-chains of the protein, an unusual pterin cofactor, Moco, is involved in both molybdenum- and tungsten-containing enzymes. The cofactor (pyranopterin-dithiolate) coordinates the metal ion via a dithiolate side-chain (Figure 17.2). In eukaryotes, the pterin side-chain has a terminal phosphate group, whereas in prokaryotes, the cofactor (R in Figure 17.2) is often a dinucleotide. 

RNA polymerase eventually reaches a transcription termination signal, at which point it will stop transcription and release the completed mRNA molecule. There are two kinds of transcription terminators commonly found in prokaryotic genes ... 

Cytochrome c and ubiquinol oxidases are part of an enzyme superfamily coupling oxidation of ferrocytochrome c (in eukaryotes) and ubiquinol (in prokaryotes) to the 4 e /4 reduction of molecular oxygen to H2O. After this introduction, we will concentrate on the cytochrome c oxidase enzyme. The two enzymes, cytochrome c oxidase (CcO) and ubiquinol oxidase, are usually defined by two criteria (1) The largest protein subunit (subunit I) possesses a high degree of primary sequence similarity across many species (2) members possess a unique bimetallic center composed of a high-spin Fe(II)/(III) heme in close proximity to a copper ion. Cytochrome c oxidase (CcO) is the terminal... 

Deactivation of an RF is feasible, however, in prokaryotes. In E. coli, three RFs are involved in the termination process, and there is a degree of codon specificity for two of them. RFl recognizes the UAG and UAA stop codons, while RF2 recognizes the UGA and UAA stop codons [16, 42]. RFS, which is not codon specific, stimulates the activity of the other two. Thus, if RF 1 is deactivated, UAA and UGA remain functional termination codons, but UAG should no longer signal for termination of protein synthesis. To test this hypothesis, the Chamberlin lab investigated a mutant strain of E. coli that produces a faulty RFl [29]. 

In eukaryotic cells, the number of initiation factors is larger and initiation is therefore more complex than in prokaryotes. The cap at the 5 end of mRNA and the polyA tail (see p. 246) play important parts in initiation. However, the elongation and termination processes are similar in all organisms. The individual steps of bacterial translation can be inhibited by antibiotics (see p. 254). 

The remaining series of reactions of fatty acid synthesis in eukary-l otes is catalyzed by the multifunctional, dimeric enzyme, fatty acid synthase. Each fatty acid synthase monomer is a multicatalytic polypeptide with seven different enzymic activities plus a domain that covalently binds a molecule of 4 -phosphopantetheine. [Note 4-Phosphopantetheine, a derivative of the vitamin pantothenic add (see p. 379), carries acetyl and acyl units on its terminal thiol (-SH)j group during fatty acid synthesis. It also is a component of 00-enzyme A.] In prokaryotes, fatty acid synthase is a multienzyme complex, and the 4 -phosphopantetheine domain is a separate protein, referred to as the acyl carrier protein (ACP). ACP is used below to refer to the phosphopantetheine-binding domain of the eukaryotic fatty acid synthase molecule. The reaction numbers in1 brackets below refer to Figure 16.9. [Note The enzyme activities listed are actually separate catalytic domains present in each mulf-1 catalytic fatty acid synthase monomer.]... 

The pathway of protein synthesis translates the three-letter alphabet of nucleotide sequences on mRNA into the twenty-letter alphabet of amino acids that constitute proteins. The mRNA is translated from its 5 -end to its 3 -end, producing a protein synthesized from its amino-terminal end to its carboxyl-terminal end. Prokaryotic mRNAs often have several coding regions, that is, they are polycistronic (see p. 420). Each coding region has its own initiation codon and produces a separate species of polypeptide. In contrast, each eukaryotic mRNA codes for only one polypeptide chain, that is, it is monocistronic. The process of translation is divided into three separate steps initiation, elongation, and termination. The polypeptide chains produced may be modified by posttranslational modification. Eukaryotic protein synthesis resembles that of prokaryotes in most details. [Note Individual differences are mentioned in the text.]... 

The unusual amino acid selenocysteine (a derivative of cysteine in which the sulfur atom is replaced by a selenium atom) is an essential component in a small number of proteins. These proteins occur in prokaryotes and eukaryotes ranging from E. coli to humans. In all cases, selenocysteine is incorporated into protein during translation in response to the codon UGA. This codon usually serves as a termination codon but occasionally, in some required but unknown context of bases, is used to specify selenocysteine instead. 

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... 

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