BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 27. PROTEIN SYNTHESIS

27.2. The Fidelity of Protein Synthesis is Determined by the High Specificity of Aminoacyl-tRNA Synthetases

Accurate Translation of genetic templates is ensured by the high Specificity of Aminoacyl-tRNA synthetases. These Enzymes are remarkably selective with respect to both the Amino Acids they activate and their cognate acceptor tRNAs. As will be discussed below, tRNA molecules that accept different amino acids possess distinct base sequences, enabling synthetases to easily recognize them. A far more formidable challenge for these enzymes is distinguishing between closely similar amino acids. For instance, the sole difference between isoleucine and valine is that isoleucine contains an extra methylene group (Fig. 27.2). The additional binding energy contributed by this extra —СН₂— group favors the activation of isoleucine at a rate roughly 200-fold greater than that of valine. Because the intracellular concentration of valine in vivo is about 5 times higher than that of isoleucine, valine would otherwise be misincorporated in place of isoleucine once in every 40 times. However, the observed error frequency in vivo is only about one in 3000. This indicates that an additional proofreading stage must exist to enhance fidelity. Indeed, the synthetase corrects its own errors. Valine erroneously activated is not transferred to isoleucine-specific tRNA. Instead, the tRNA promotes the Hydrolysis of valyl-AMP, thereby preventing its erroneous incorporation into Proteins. Furthermore, this hydrolytic reaction frees the synthetase to activate and transfer the correct amino acid, isoleucine. How does the synthetase avoid hydrolyzing isoleucyl-AMP, the correct intermediate? Most likely, the hydrolytic site is large enough to accommodate valyl-AMP, but too small to bind isoleucyl-AMP.

Class="center">Fig. 27.2. Space-filling models of valine and isoleucine. Synthetases active toward these Amino acids are highly specific

Fig. 27.3. Error correction via hydrolysis of the erroneous product

Many other aminoacyl-tRNA synthetases also contain hydrolytic sites In addition to their synthetic sites. These sites likely act as double filters, ensuring exceptional fidelity. The synthetic site rejects amino acids larger than the required one, whereas the hydrolytic site destroys activated intermediates that are smaller than required. Evidently, the high fidelity of Protein Synthesis depends primarily on a correction mechanism driven by the hydrolytic activity possessed by many aminoacyl-tRNA synthetases.

27.3. Transfer RNA Molecules Share a Common Structural Plan

In 1965, after seven years of persistent research, Robert Holley became the first to determine The base sequence of a Transfer RNA molecule. The elucidation of Yeast Alanine tRNA provided the first complete nucleic acid sequence, sparking new insights into the biological activity of tRNA molecules. In the course of decoding this sequence, Holley developed general Methods for determining nucleotide sequences in Nucleic Acids. The sequence of yeast alanine tRNA is shown in Fig. 27.4. The molecule consists of a single chain of 76 ribonucleotides. The 5'-end is phosphorylated (pG), and the 3'-end possesses a free 3'-hydroxyl group. A hallmark of this molecule is the high content of bases other than A, U, G, and C. It contains nine unusual NUCLEOTIDES: inosine, pseudouridine, dihydrouridine, ribothymidine, and methylated derivatives of guanosine and inosine. The amino acid attachment site is the 3'-hydroxyl group of the terminal adenosine residue at the 3'-end of the molecule. The IGC sequence in the middle of the molecule serves as the anticodon, which is complementary to GCC—one of the codons for alanine.

Fig. 27.4. Base sequence of yeast alanine tRNA. Modified nucleosides (highlighted in green) are abbreviated as follows: inosine — I, methylinosine — mI, dihydrouridine — UH₂, ribothymidine — T, pseudouridine — , methylguanosine — mG, and dimethylguanosine — m2G

The sequences of several other tRNA molecules were soon elucidated. To date, the sequences of more than 70 tRNAs are known. Most remarkably, all these sequences can be folded into a cloverleaf Secondary Structure in which approximately half of the nucleotides are base-paired. Consequently, tRNA molecules share numerous structural features. This is hardly unexpected, since

all tRNA molecules must interact in a nearly identical manner with Ribosomes and mRNA. Specifically, every tRNA must fit into the A and P sites of the ribosome and interact with the enzyme that catalyzes peptide bond formation.

All transfer RNA molecules share the following General Properties.

1. They are all single chains ranging from 73 to 93 ribonucleotides in length (molecular mass of approximately 25 kDa).

2. They contain many unusual bases, typically 7 to 15 per molecule. Many of these modified bases are methylated or dimethylated derivatives of A, U, C, and G, formed via the enzymatic modification of a precursor tRNA (Section 25.17). The Physiological Role of these unusual bases remains unknown. It is quite possible that methylation prevents certain base pairings, thereby rendering those regions accessible for other interactions. Furthermore, methylation alters the Hydrophobicity of specific segments of tRNA, which may be crucial for their interaction with synthetases and ribosomal proteins.

3. The 5'-end of tRNA is phosphorylated, typically bearing a pG residue.

4. The 3'-end of all tRNAs contains a CCA sequence. The activated amino acid is attached to the 3'-hydroxyl group of the terminal adenosine.

5. Roughly half of the nucleotides in tRNA are base-paired to form double-helical regions (Fig. 27.5). Five groups of bases remain unpaired: the 3'-terminal CCA region (acceptor stem); the TC loop, named for its characteristic ribothymidine-pseudouracil-cytosine sequence; the variable loop (or extra arm), which varies in size among different tRNAs; the dihydrouridine loop, which contains several dihydrouridine residues; and the anticodon loop.

6. The anticodon loop consists of seven bases arranged in the following sequence:

Fig. 27.5. General structural scheme of tRNA molecules

27.4. Transfer RNA adopts an L-shaped conformation

To date, the three-dimensional STRUCTURE OF THE tRNA molecule has been resolved at the atomic level thanks to X-ray crystallographic studies carried out in the laboratories of Alexander Rich and Aaron Klug. Their independent X-Ray Diffraction analyses of phenylalanine tRNA yielded a wealth of new insights into tRNA Structure.

1. The molecule has an L-shape (Figs. 27.6 and 27.7).

Fig. 27.6. Photograph of the skeletal model of yeast phenylalanine tRNA, based on an electron density map at 3 Å resolution

Fig. 27.7. Schematic representation of the three-dimensional structure of yeast phenylalanine tRNA

2. The molecule contains two double-helical regions. Each of these helices contains approximately ten Base Pairs, corresponding to one turn of the helix. The helical segments are oriented perpendicularly to each other, which gives the molecule its L-shape. The base-pairing scheme postulated in the cloverleaf model, which was derived from sequence determinations, proved to be correct.

3. Most of the bases outside the helical regions form unconventional Hydrogen Bonds. These tertiary interactions occur between bases that are not typically complementary (e.g., G—G, A—A, and A—C). Furthermore, the ribose-phosphate backbone interacts with certain bases and even with another segment of the backbone itself. In many such interactions, the 2'-OH group of ribose residues acts as a Hydrogen bond donor or acceptor. In addition, a significant proportion of the bases are base-stacked (interplanar interactions). These hydrophobic interactions between neighboring aromatic rings play a crucial role in molecular architecture.

4. The CCA terminus—the amino acid attachment site—is located at one end of the L. The other end of the L corresponds to the anticodon loop. Thus, the amino acid attached to aminoacyl-tRNA is separated from the anticodon by a distance of roughly 80 Å. The dihydrouridine loop and the TC loop form the corner of the L.

5. The CCA terminus and the adjacent helical region do not interact very strongly with the rest of the molecule. This portion of the molecule can undergo conformational changes during Amino Acid Activation and protein synthesis on the ribosome.

Determining the three-dimensional structure of tRNA marks a major milestone in elucidating The Mechanism of translation at THE MOLECULAR LEVEL. Until recently, solving this problem seemed a distant prospect. Today, researchers' ideas and efforts in this field are shifting toward even more complex challenges, such as determining the three-dimensional structures of aminoacyl-tRNA synthetase–tRNA complexes, as well as tRNA bound to mRNA and ribosomal proteins.