Ribosomes prokaryotes

The charging of the tRNA molecule with the aminoacyl moiety requires the hydrolysis of an ATP to an AMP, equivalent to the hydrolysis of two ATPs to two ADPs and phosphates. The entry of the aminoacyl-tRNA into the A site results in the hydrolysis of one GTP to GDP. Translocation of the newly formed pep-tidyl-tRNA in the A site into the P site by EF2 similarly results in hydrolysis of GTP to GDP and phosphate. Thus, the energy requirements for the formation of one peptide bond include the equivalent of the hydrolysis of two ATP molecules to ADP and of two GTP molecules to GDP, or the hydrolysis of four high-energy phosphate bonds. A eukaryotic ribosome can incorporate as many as six amino acids per second prokaryotic ribosomes incorporate as many as 18 per second. Thus, the process of peptide synthesis occurs with great speed and accuracy until a termination codon is reached. 

Gribskov, M. (1992). Translational initiation factors IF-1 and eIF-2 alpha share an RNA-binding motif with prokaryotic ribosomal protein SI and polynucleotide phosphoryl-ase. Gene 119, 107-111. 

Ribosomes are found in all organisms and consist of two unequal subunits. Prokaryotic ribosomes (e.g., those from Escherichia coli) are the most widely studied and those with which this article will be primarily concerned. The 50 S subunit of the E. coli ribosome consists of one strand each of 5 S and 23 S rRNA, and 32 different proteins. The 30 S subunit contains 16 S rRNA and 21 different proteins. 

Two-dimensional periodic organization of eukaryotic and prokaryotic ribosomes occurs under special conditions in vivo (Byers, 1967 Kress et al., 1971 Taddei, 1972 O Brien et al, 1980) or in vitro (Barbieri, 1979 Clark et al, 1982). The two-dimensional sheets have been analyzed by image-reconstruction techniques (Kiihlbrandt and Unwin, 1982 Clark et al, 1982). 

Rjbosomal Proteins Their Structure and Spatial Arrangement in Prokaryotic Ribosomes... 

The large and small prokaryotic ribosomal subunits are SOS and 30S, respectively. The complete prokaryotic ribosome is a 70S particle. (Note The S values are determined by behavior of the particles in an ultracentrifuge. They are a function of both size and shape, and therefore the numbers are not additive.)... 

Certain antibiotics (for example, chloramphenicol) inhibit mitochondrial protein synthesis, but not cytoplasmic protein synthesis, because mitochondrial ribosomes are similar to prokaryotic ribosomes. 

The arrangement of the individual components of a ribosome has now been determined for prokaryotic ribosomes. It is known that filamentous mRNA passes through a cleft between the two subunits near the characteristic horn on the small subunit. tRNAs also bind near this site. The illustration shows the size of a tRNA molecule for comparison. 

Prokaryotic ribosomes have a similar structure, but are somewhat smaller than those of eukaryotes (sedimentation coef cient 70 S for the complete ribosome, 30 S and 50 S for the subunits). Mitochondrial and chloroplast ribosomes are comparable to prokaryotic ones. 

Prokaryotic ribosomes contain three rRNAs 16S rRNA in the small (30S) subunit and 23S and 5S rRNA molecules in the large (50S) subunit. 

Many of the inhibitors of protein synthesis are selective for prokaryotic ribosomes, which reduces potential for toxicity to humans. 

Tetracycline and its derivatives Inhibit entry of the aminoacyl-tRNAs into the A site of both eukaryotic and prokaryotic ribosomes, but eukaryotic plasma membranes are impermeable to these drugs. 

Ribosome recycling factor (RRF) and elongation factor-G (EF-G) are required to recycle the prokaryotic ribosome back to a new round of initiation after termination (Nakamura and Ito 2003). No recycling factor has been identified so far in the cytoplasm of eukaryotic cells. To explain this difference it has been postulated that eukaryotic eRF3 has a dual function ... 

Lynch SR, Puglisi JD (2001) Structural origins of aminoglycoside specificity for prokaryotic ribosomes. J... 

Ribosomal RNA (rRNA) As discussed on p. 414, prokaryotic ribosomes contain three molecules of rRNA, whereas eukaryotic ribosomes contain four molecules of rRNA (see Figure 31.8). The rRNAs have extensive regions of secondary structure arising from the base-pairing of complementary sequences of nucleotides in different portions of the molecule. The formation of intramolecular, double-stranded regions is comparable to that found in tRNA. 

Ribosomal proteins Ribosomal proteins are present in considerably greater numbers in eukaryotic ribosomes than in prokaryotic ribosomes. These proteins play a number of roles in the structure and function of the ribosome and its interactions with other components of the translation system. 

The importance of the Shine-Dalgarno sequence is underscored by the action of the bacterial toxin colecin E3. This toxin inactivates the small subunit of the prokaryotic ribosome by cleavage of about 50 residues from the 3 terminus of 16S rRNA. The cleavage disrupts the sequence that is complementary to the Shine-Dalgarno sequence and thus specifically inhibits the initiation process. Because of the fundamental differences between prokaryotic and eukaryotic initiation just described, colecin E3 does not inhibit the eukaryotic ribosome. 

Eukaryotic ribosomes are larger (80S) and more complex than prokaryotic ribosomes (70S). Initiation is basically similar in prokaryotes and eukaryotes except that in eukaryotes at least nine initiation factors are involved (cf. three factors in prokaryotes), the initiating amino acid is methionine (cf. N-formylmethionine in prokaryotes), eukaryotic mRNAs do not contain Shine-Dalgarno sequences (so the AUG initiation codon is detected by the ribosome scanning instead), and eukaryotic mRNA is monocistronic (cf. some polycistronic mRNAs in prokaryotes). Initiation in eukaryotes involves the formation of a 48S preinitiation complex between the 40S ribosomal subunit, mRNA, initiation factors and Met-tRNA 61. The ribosome then scans the mRNA to locate the AUG initiation codon. The 60S ribosomal subunit now binds to form the 80S initation complex. 

Whereas a prokaryotic ribosome has a sedimentation coefficient (see Topic G9) of 70S and subunits of 30S and 50S, a eukaryotic ribosome has a sedimentation coefficient of 80S with subunits of 40S and 60S (see Topic G9). The composition of eukaryotic ribosomal subunits is also more complex than prokaryotic subunits (see Topic G9) but the function of each subunit is essentially the same as in prokaryotes. 

The steps of initiation occur on the isolated small subunit (30S) of the prokaryotic ribosome. Ribosomes contain two subunits, a 30S and 50S subunit, which associate to form a 70S particle. (The S values refer to the rate at which each component sediments in the ultracentrifuge they don t always add up.) In general, the 30S subunit is mostly involved in the decoding and tRNA-mRNA interaction process, while the 50S subunit is involved in actual peptide bond synthesis. Ribosomal subunits are dissociated prior to the initiation reaction. 

The features of the ribosome display construct are summarized in Figure 3. On the DNA level, the construct requires a T7 promoter for efficient in vitro transcription to mRNA. On the mRNA level, the construct contains, as a regulatory sequence for translation, either a prokaryotic ribosome binding site (Shine and Dalgamo, 1975) if the E. coli... 

Prokaryotic ribosomes attach to the nascent mRNA while it is still being transcribed. Because transcription and translation are coupled, prokaryotic mRNAs undergo little modification and processing before being used as templates for protein synthesis. Prokaryotic tRNA and rRNA are transcribed in units larger than those ultimately used and must be processed to generate the functional molecules. The processing of these and the eukaryotic primary transcripts, almost all of which require modification, is discussed in a later section. 

The three antibiotic inhibitors of translation that will be used in this experiment are chloramphenicol, cycloheximide, and puromycin (Fig. 23-10). Chloramphenicol is specific for prokaryotic ribosomes, blocking the transfer of the peptide on the tRNA at the P site to the amino acid linked to the tRNA at the A site (the peptidyl transfer reaction). Since the source of the ribosomes used in this experiment is wheat germ (eukaryotic), we would predict that chloramphenicol would not have a great effect on translation. The mechanism of cycloheximide-mediated inhibition is the same as that described above for chloramphenicol, except for the fact that it is specific for the 80S eukaryotic ribosome. Puromycin is a more broad translational inhibitor, effective on both eukaryotic and prokaryotic ribosomes. It acts as a substrate analog of aminoacyl tRNA. When it binds at the A site of the ribosome, it induces premature termination of translation (Fig. 

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