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stryer

Biochemistry

5th

Jeremy M Berg,1 John L Tymoczko,2 and Lubert Stryer3

1Johns Hopkins University School of Medicine

2Carleton College

3Stanford University

W. H. Freeman and Company0-7167-3051-02002

biochemistry

Chapter 29: 29.1 Protein Synthesis Requires the Translation of Nucleotide Sequences Into Amino Acid Sequences

Figure 29.2
Polypeptide-Chain Growth

An external file that holds a picture, illustration, etc., usually as some form of binary object. The name of referred object is ch29f2.jpg.

Figure 29.2

Polypeptide-Chain Growth

Proteins are synthesized by the successive addition of amino acids to the carboxyl terminus.

The basics of protein synthesis are the same across all kingdoms of life, attesting to the fact that the protein-synthesis system arose very early in evolution. A protein is synthesized in the amino-to-carboxyl direction by the sequential addition of amino acids to the carboxyl end of the growing peptide chain (Figure 29.2

). The amino acids arrive at the growing chain in activated form as aminoacyl-tRNAs, created by joining the carboxyl group of an amino acid to the 3′ end of a transfer RNA molecule. The linking of an amino acid to its corresponding tRNA is catalyzed by an aminoacyl-tRNA synthetase. ATP cleavage drives this activation reaction. For each amino acid, there is usually one activating enzyme and at least one kind of tRNA.

29.1.1. The Synthesis of Long Proteins Requires a Low Error Frequency

The process of transcription is analogous to copying, word for word, a page from a book. There is no change of alphabet or vocabulary; so the likelihood of a change in meaning is small. Translating the base sequence of an mRNA molecule into a sequence of amino acids is similar to translating the page of a book into another language. Translation is a complex process, entailing many steps and dozens of molecules. The potential for error exists at each step. The complexity of translation creates a conflict between two requirements: the process must be not only accurate, but also fast enough to meet a cell's needs. How fast is “fast enough”? In E.coli, translation takes place at a rate of 40 amino acids per second, a truly impressive speed considering the complexity of the process.

How accurate must protein synthesis be? Let us consider error rates. The probability p of forming a protein with no errors depends on n, the number of amino acid residues, and ϵ, the frequency of insertion of a wrong amino acid:

Table 29.1
Accuracy of protein synthesis

Table 29.1

Accuracy of protein synthesis

	Probability of synthesizing an error-free protein
	Number of amino acid residues
Frequency of inserting an incorrect amino acid	100	300	1000
10^-2	0.366	0.049	0.000
10^-3	0.905	0.741	0.368
10^-4	0.990	0.970	0.905
10^-5	0.999	0.997	0.990

As Table 29.1 shows, an error frequency of 10^-2 would be intolerable, even for quite small proteins. An ϵ value of 10^-3 would usually lead to the error-free synthesis of a 300-residue protein (~33 kd) but not of a 1000-residue protein (~110 kd). Thus, the error frequency must not exceed approximately 10^-4 to produce the larger proteins effectively. Lower error frequencies are conceivable; however, except for the largest proteins, they will not dramatically increase the percentage of proteins with accurate sequences. In addition, such lower error rates are likely to be possible only by a reduction in the rate of protein synthesis because additional time for proofreading will be required. In fact, the observed values of ϵ are close to 10^-4. An error frequency of about 10^-4 per amino acid residue was selected in the course of evolution to accurately produce proteins consisting of as many as 1000 amino acids while maintaining a remarkably rapid rate for protein synthesis.

29.1.2. Transfer RNA Molecules Have a Common Design

The fidelity of protein synthesis requires the accurate recognition of three-base codons on messenger RNA. Recall that the genetic code relates each amino acid to a three-letter codon (Section 5.5.1). An amino acid cannot itself recognize a codon. Consequently, an amino acid is attached to a specific tRNA molecule that can recognize the codon by Watson-Crick base pairing. Transfer RNA serves as the adapter molecule that binds to a specific codon and brings with it an amino acid for incorporation into the polypeptide chain.

Figure 29.3
Alanine-tRNA Sequence

An external file that holds a picture, illustration, etc., usually as some form of binary object. The name of referred object is ch29f3.jpg.

Figure 29.3

Alanine-tRNA Sequence

The base sequence of yeast alanyl-tRNA and the deduced cloverleaf secondary structure are shown. Modified nucleosides are abbreviated as follows: methylinosine (mI), dihydrouridine (UH₂), ribothymidine (T), pseudouridine (Ψ), methylguanosine (mG), and dimethylguanosine (m₂G). Inosine (I), another modified nucleoside, is part of the anticodon.

Robert Holley first determined the base sequence of a tRNA molecule in 1965, as the culmination of 7 years of effort. Indeed, his study of yeast alanyl-tRNA provided the first complete sequence of any nucleic acid. This adapter molecule is a single chain of 76 ribonucleotides (Figure 29.3

). The 5′ terminus is phosphorylated (pG), whereas the 3′ terminus has a free hydroxyl group. The amino acid attachment site is the 3′-hydroxyl group of the adenosine residue at the 3′ terminus of the molecule. The sequence IGC in the middle of the molecule is the anticodon. It is complementary to GCC, one of the codons for alanine.

Figure 29.4
General Structure of tRNA Molecules

An external file that holds a picture, illustration, etc., usually as some form of binary object. The name of referred object is ch29f4.jpg.

Figure 29.4

General Structure of tRNA Molecules

Comparison of the base sequences of many tRNAs reveals a number of conserved features.

The sequences of several other tRNA molecules were determined a short time later. Hundreds of sequences are now known. The striking finding is that all of them can be arranged in a cloverleaf pattern in which about half the residues are base-paired (Figure 29.4

). Hence, tRNA molecules have many common structural features. This finding is not unexpected, because all tRNA molecules must be able to interact in nearly the same way with the ribosomes, mRNAs, and protein factors that participate in translation.

All known transfer RNA molecules have the following features:

1
Each is a single chain containing between 73 and 93 ribonucleotides (~25 kd).
2
They contain many unusual bases, typically between 7 and 15 per molecule. Some are methylated or dimethylated derivatives of A, U, C, and G formed by enzymatic modification of a precursor tRNA (Section 28.1.8). Methylation prevents the formation of certain base pairs, thereby rendering some of the bases accessible for other interactions. In addition, methylation imparts a hydrophobic character to some regions of tRNAs, which may be important for their interaction with synthetases and ribosomal proteins. Other modifications alter codon recognition, as will be discussed shortly.
3
About half the nucleotides in tRNAs are base-paired to form double helices. Five groups of bases are not base paired in this way: the 3′ CCA terminal region, which is part of a region called the acceptor stem; the TψC loop, which acquired its name from the sequence ribothymine-pseudouracil-cytosine; the “extra arm,” which contains a variable number of residues; the DHU loop, which contains several dihydrouracil residues; and the anticodon loop. The structural diversity generated by this combination of helices and loops containing modified bases ensures that the tRNAs can be uniquely distinguished, though structurally similar overall.
4
The 5′ end of a tRNA is phosphorylated. The 5′ terminal residue is usually pG.
5
The activated amino acid is attached to a hydroxyl group of the adenosine residue located at the end of the 3′ CCA component of the acceptor stem. This region is single stranded at the 3′ end of mature rRNAs.
6
The anticodon is present in a loop near the center of the sequence.

29.1.3. The Activated Amino Acid and the Anticodon of tRNA Are at Opposite Ends of the L-Shaped Molecule

The three-dimensional structure of a tRNA molecule was first determined in 1974 through x-ray crystallographic studies carried out in the laboratories of Alexander Rich and Aaron Klug. The structure determined, that of yeast phenylalanyl-tRNA, is highly similar to all structures subsequently determined for other tRNA molecules. The most important properties of the tRNA structure are:

1

Figure 29.5
L-Shaped tRNA Structure

Figure 29.5
.

L-Shaped tRNA Structure
A skeletal model of yeast phenylalanyl-tRNA reveals the L-shaped structure. The CCA region is at the end of one arm, and the anticodon loop is at the end of the other.

The molecule is L-shaped (Figure 29.5).
2

Figure 29.6
Helix Stacking in tRNA

Figure 29.6
.

Helix Stacking in tRNA

The four helices of the secondary structure of tRNA (see Figure 29.4) stack to form an L-shaped structure.

There are two apparently continuous segments of double helix. These segments are like A-form DNA, as expected for an RNA helix (Section 27.1.1). The base-pairing predicted from the sequence analysis is correct. The helix containing the 5′ and 3′ ends stacks on top of the helix that ends in the TψC loop to form one arm of the L; the remaining two helices stack to form the other (Figure 29.6).
3
Most of the bases in the nonhelical regions participate in hydrogenbonding interactions, even if the interactions are not like those in Watson-Crick base pairs.
4
The CCA terminus containing the amino acid attachment site extends from one end of the L. This single-stranded region can change conformation during amino acid activation and protein synthesis.
5
The anticodon loop is at the other end of the L, making accessible the three bases that make up the anticodon.

Thus, the architecture of the tRNA molecule is well suited to its role as adaptor; the anticodon is available to interact with an appropriate codon on mRNA while the end that is linked to an activated amino acid is well positioned to participate in peptide-bond formation.

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