Ribosome tRNA-binding sites

Several key concepts are worth remembering. GTP is used as an energy source for translation, but ATP is used to form the aminoacyl-tRNA. The ribosome effectively has two kinds of tRNA binding sites. Only tRNAMet can bind to the P (for peptide) site, and this only occurs during the initial formation of the functional ribosome (initiation). All other aminoacyl-tRNAs enter at the A (for amino acid) binding site. After formation of the peptide bond (this doesn t require GTP hydrolysis), the tRNA with the growing peptide attached is moved (translocated) to the other site (this does require GTP hydrolysis). 

In the 70 S initiation complex, formylme-thionine tRNA is initially located at a binding site known as the peptidyl site (P). A second binding site, the acceptor site (A), is not yet occupied during this phase of translation. Sometimes, a third tRNA binding site is defined as an exit site (E), from which uncharged tRNAs leave the ribosome again (see p. 252 not shown). 

Several sites on the ribosome interact with tRNA (Fig. 4.1) and also are targeted by antibiotics. The peptidyl-tRNA binding site (P-site) of the large subunit binds to the 3 end of peptidyl-tRNA. The aminoacyl-tRNA binding site (A-site) of the... 

Electron microscopic studies of the ribosome at increasingly high resolution provided views of the overall structure and revealed the positions of tRNA-binding sites. Astounding progress on the structure of the ribosome has been made by x-ray crystallographic methods, after the pioneering work by Ada Yonath. The structures of both the 30S and the 50S subunits have been determined at or close to atomic resolution, and the elucidation of the structure of intact 70S... 

Ribosomes Have Three tRNA-Binding Sites That Bridge the 30S and SOS Subunits... 

Figure 29.22. Transfer RNA-Binding Sites. (A) Three tRNA-binding sites are present on the 70S ribosome. They are called the A (for aminoacyl), P (for peptidyl), and E (for exit) sites. Each tRNA molecule contacts both the SOS and the 50S subunit. (B) The tRNA molecules in sites A and P are base paired with mRNA.

Tagging a ribosomal site. Design an affinity-labeling reagent for one of the tRNA binding sites in E. coli ribosomes. 

Ribosomal RNA (rRNA) is a component of the ribosomes, the protein synthesis factories in the cell. rRNA molecules are extremely abundant, making up at least 80 percent of the RNA molecules found in a typical eukaryotic cell. Virtually all ribosomal proteins are in contact with rRNA. Most of the contacts between ribosomal subunits are made between the 16S and 23 S rRNAs such that the interactions involving rRNA are a key part of ribosome function. The environment of the tRNA-binding sites is largely determined by rRNA. The rRNA molecules have several roles in protein synthesis. 16S rRNA plays an active role in the functions of the 308 subunit. It interacts directly with mRNA, with the 508 subunit, and with the anticodons of tRNAs in the P- and A-sites. Peptidyl transferase activity resides exclusively in the 238 rRNA. Finally, the rRNA molecules have a structural role. They fold into three-dimensional shapes that form the scaffold on which the ribosomal proteins assemble. 

The ribosome has two sites for binding tRNA molecules. The first site, called the peptidyl tRNA binding site (P-site), holds the peptidyl tRNA, the growing peptide bound to a tRNA molecule. The second site, called the aminoacyl tRNA... 

Three tRNA binding sites on the 70S ribosome are involved in the process of elongation. Figure 27.22 shows a single round of this cycle, which is repeated until a signal for termination is encountered. The cycle goes as follows ... 

Figure 1.84 Schematic of translation. The mRNA codons are read and converted from nucleoside sequences to protein primary structure by means of cognate aminoacyl-tRNAs. All mRNA codons are translated at a ribosome (prepared from rRNA) that has two cognate aminoacyl-tRNA binding sites P (peptidyl) and A (aminoacyl). All tRNAs are "adaptors" that can bind a particular mRNA codon through their anticodon loop, using Watson-Crick base pairing, and also associate covalently with the appropriate amino acid residue coded for by the corresponding mRNA codon When two cognate aminoacyl-tRNA molecules bind mRNA in P and A sites (a), then both are close enough for peptide link formation to take place with the emergence of a peptide chain (b). As amino acyl tRNA molecules continue to dock sequentially onto mRNA codons (in the direction 5 (c), and amino acid residues continue to be added (W —> C ) (d),...

Ribosomes (Fig. 69.1) contain three tRNA binding sites called E-site, P-site and A-site which are involved in protein synthesis (Chapter 70). 

Because the sequence T- F-C-Cp blocks aminoacyl tRNA binding sites to ribosomes more effectively than any other similar sequence, it is believed that T- F-C-Cp is part of the nucleotide sequence which binds to ribosomes. This conclusion needs, however, to be reconciled with the fact that eukaryotic initiator meth-ionyl-tRNA of yeast and rabbit lacks the sequence G-T- F-C-G(A-) which is replaced by G-A-U-C-G [248-249]. 

In a considerable number of cases, peptidyl-tRNA affinity probes were shown to modify primarily not the ribosomal proteins, but the 23 S rRNA. Further characterization of modified sites was obtained by splitting of the 23 S rRNA into 13 S and 18 S fragments including the 5 third and the 3 two-thirds of the molecule, respectively. Mapping of tRNA Binding Sites. Identification of ribosomal components interacting with parts of tRNA other than the 3 end has been attempted by preparing tRNAs derivatized in odd bases with photosensitive residues. Affinity probes are listed in Table III. Assays for func-... 

Although not emphasized in this chapter, it should be clear that exactly the same approach can be applied to any other tRNA binding site such as exists in AA-tRNA synthetases, in elongation factors, and in eukaryotic ribosomes. Some studies on Phe-tRNA synthetase of Escherichia coli- are illustrative of this approach. 

Fio. 1. Scheme of peptideribosomal subunit. The large oval shape represents the 50S ribosomal subunit. A and P indicated in die 50S subunit stand for hypothetical tRNA binding sites. The symbols n—1, n, n + 1, n + 2, and n -f- 3 represent a series of adjacent codons in a segment of mRNA rt represents a tRNA molecule. 

The subcellular organelle concerned with protein synthesis is the ribosome. This consists of two subunits, composed of RNA with a variety of associated proteins. The ribosome permits the binding of the anticodon region of amino acyl tRNA to the codon on mRNA, and aligns the amino acids for formation of peptide bonds. As shown in Figure 9.6, the ribosome binds to mRNA, and has two tRNA binding sites. One, the P site, contains the growing peptide chain, attached to tRNA, while the other, the A site, binds the next amino acyl tRNA to be incorporated into the peptide chain. 

The association of ribosomal subunits produces two distinguishable sites (called P for peptidyl-tRNA binding site and A for aminoacyl-tRNA binding site) which function in the addition of amino acids to the formylmethionyl residue. According to the current two-site model (Figure 17.8), the 70S initiation complex contains an occupied site P. The appropriate ternary complex, con-... 

It has been known for some time that tetracyclines are accumulated by bacteria and prevent bacterial protein synthesis (Fig. 4). Furthermore, inhibition of protein synthesis is responsible for the bacteriostatic effect (85). Inhibition of protein synthesis results primarily from dismption of codon-anticodon interaction between tRNA and mRNA so that binding of aminoacyl-tRNA to the ribosomal acceptor (A) site is prevented (85). The precise mechanism is not understood. However, inhibition is likely to result from interaction of the tetracyclines with the 30S ribosomal subunit because these antibiotics are known to bind strongly to a single site on the 30S subunit (85). 

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