By convention, codons are always more This genetic code is used universally in all present-day organisms. Although a few slight differences in the code have been found, these are chiefly in the DNA of mitochondria.
Mitochondria have their own transcription and protein synthesis systems that operate quite independently from those of the rest of the cell, and it is understandable that their small genomes have been able to accommodate minor changes to the code discussed in Chapter In principle, an RNA sequence can be translated in any one of three different reading frames , depending on where the decoding process begins Figure However, only one of the three possible reading frames in an mRNA encodes the required protein.
We see later how a special punctuation signal at the beginning of each RNA message sets the correct reading frame at the start of protein synthesis. The three possible reading frames in protein synthesis. The codons in an mRNA molecule do not directly recognize the amino acids they specify: the group of three nucleotides does not, for example, bind directly to the amino acid.
Rather, the translation of mRNA into protein depends on adaptor molecules that can recognize and bind both to the codon and, at another site on their surface, to the amino acid. We saw earlier in this chapter that RNA molecules can fold up into precisely defined three-dimensional structures, and the tRNA molecules provide a striking example.
Four short segments of the folded tRNA are double-helical, producing a molecule that looks like a cloverleaf when drawn schematically Figure A. The cloverleaf undergoes further folding to form a compact L-shaped structure that is held together by additional hydrogen bonds between different regions of the molecule Figure B , C. A tRNA molecule. In this series of diagrams, the same tRNA molecule—in this case a tRNA specific for the amino acid phenylalanine Phe —is depicted in various ways.
A The cloverleaf structure, a convention used to show the complementary more Two regions of unpaired nucleotides situated at either end of the L-shaped molecule are crucial to the function of tRNA in protein synthesis. One of these regions forms the anticodon , a set of three consecutive nucleotides that pairs with the complementary codon in an mRNA molecule.
We have seen in the previous section that the genetic code is redundant; that is, several different codons can specify a single amino acid see Figure This redundancy implies either that there is more than one tRNA for many of the amino acids or that some tRNA molecules can base -pair with more than one codon.
In fact, both situations occur. Some amino acids have more than one tRNA and some tRNAs are constructed so that they require accurate base-pairing only at the first two positions of the codon and can tolerate a mismatch or wobble at the third position Figure This wobble base-pairing explains why so many of the alternative codons for an amino acid differ only in their third nucleotide see Figure In bacteria, wobble base-pairings make it possible to fit the 20 amino acids to their 61 codons with as few as 31 kinds of tRNA molecules.
The exact number of different kinds of tRNAs, however, differs from one species to the next. For example, humans have tRNA genes but, among them, only 48 different anticodons are represented.
Wobble base-pairing between codons and anticodons. If the nucleotide listed in the first column is present at the third, or wobble, position of the codon, it can base-pair with any of the nucleotides listed in the second column. Thus, for example, when more We have seen that most eucaryotic RNAs are covalently altered before they are allowed to exit from the nucleus , and tRNAs are no exception. In addition, some tRNA precursors from both bacteria and eucaryotes contain introns that must be spliced out.
This splicing reaction is chemically distinct from that of pre- mRNA splicing; rather than generating a lariat intermediate, tRNA splicing occurs through a cut-and-paste mechanism that is catalyzed by proteins Figure Trimming and splicing both require the precursor tRNA to be correctly folded in its cloverleaf configuration.
Because misfolded tRNA precursors will not be processed properly, the trimming and splicing reactions are thought to act as quality-control steps in the generation of tRNAs. The endonuclease a four-subunit enzyme removes the tRNA intron blue. Courtesy more All tRNAs are also subject to a variety of chemical modifications—nearly one in 10 nucleotides in each mature tRNA molecule is an altered version of a standard G , U, C, or A ribonucleotide.
Over 50 different types of tRNA modifications are known; a few are shown in Figure Some of the modified nucleotides—most notably inosine, produced by the deamination of guanosine—affect the conformation and base -pairing of the anticodon and thereby facilitate the recognition of the appropriate mRNA codon by the tRNA molecule see Figure Others affect the accuracy with which the tRNA is attached to the correct amino acid.
A few of the unusual nucleotides found in tRNA molecules. These nucleotides are produced by covalent modification of a normal nucleotide after it has been incorporated into an RNA chain. We now consider how each tRNA molecule becomes linked to the one amino acid in 20 that is its appropriate partner. Recognition and attachment of the correct amino acid depends on enzymes called aminoacyl-tRNA synthetases , which covalently couple each amino acid to its appropriate set of tRNA molecules Figures and For most cells there is a different synthetase enzyme for each amino acid that is, 20 synthetases in all ; one attaches glycine to all tRNAs that recognize codons for glycine, another attaches alanine to all tRNAs that recognize codons for alanine, and so on.
Many bacteria, however, have fewer than 20 synthetases, and the same synthetase enzyme is responsible for coupling more than one amino acid to the appropriate tRNAs. In these cases, a single synthetase places the identical amino acid on two different types of tRNAs, only one of which has an anticodon that matches the amino acid. Amino acid activation. The two-step process in which an amino acid with its side chain denoted by R is activated for protein synthesis by an aminoacyl-tRNA synthetase enzyme is shown.
As indicated, the energy of ATP hydrolysis is used to attach each more The structure of the aminoacyl-tRNA linkage. The carboxyl end of the amino acid forms an ester bond to ribose.
Because the hydrolysis of this ester bond is associated with a large favorable change in free energy, an amino acid held in this way is said more The energy of this bond is used at a later stage in protein synthesis to link the amino acid covalently to the growing polypeptide chain.
Although the tRNA molecules serve as the final adaptors in converting nucleotide sequences into amino acid sequences, the aminoacyl-tRNA synthetase enzymes are adaptors of equal importance in the decoding process Figure This was established by an ingenious experiment in which an amino acid cysteine was chemically converted into a different amino acid alanine after it already had been attached to its specific tRNA. Although cells have several quality control mechanisms to avoid this type of mishap, the experiment clearly establishes that the genetic code is translated by two sets of adaptors that act sequentially.
Each matches one molecular surface to another with great specificity, and it is their combined action that associates each sequence of three nucleotides in the mRNA mole -cule—that is, each codon —with its particular amino acid. The genetic code is translated by means of two adaptors that act one after another. The first adaptor is the aminoacyl-tRNA synthetase, which couples a particular amino acid to its corresponding tRNA; the second adaptor is the tRNA molecule itself, whose more The synthetase must first select the correct amino acid, and most do so by a two-step mechanism.
First, the correct amino acid has the highest affinity for the active-site pocket of its synthetase and is therefore favored over the other In particular, amino acids larger than the correct one are effectively excluded from the active site.
However, accurate discrimination between two similar amino acids, such as isoleucine and valine which differ by only a methyl group , is very difficult to achieve by a one-step recognition mechanism. A second discrimination step occurs after the amino acid has been covalently linked to AMP see Figure When tRNA binds the synthetase, it forces the amino acid into a second pocket in the synthetase, the precise dimensions of which exclude the correct amino acid but allow access by closely related amino acids.
Once an amino acid enters this editing pocket, it is hydrolyzed from the AMP or from the tRNA itself if the aminoacyl-tRNA bond has already formed and released from the enzyme.
This hydrolytic editing, which is analogous to the editing by DNA polymerases Figure , raises the overall accuracy of tRNA charging to approximately one mistake in 40, couplings. Hydrolytic editing. A tRNA synthetases remove their own coupling errors through hydrolytic editing of incorrectly attached amino acids.
As described in the text, the correct amino acid is rejected by the editing site. B The error-correction process more Most tRNA synthetases directly recognize the matching tRNA anticodon ; these synthetases contain three adjacent nucleotide -binding pockets, each of which is complementary in shape and charge to the nucleotide in the anticodon.
For other synthetases it is the nucleotide sequence of the acceptor stem that is the key recognition determinant. Having seen that amino acids are first coupled to tRNA molecules, we now turn to the mechanism by which they are joined together to form proteins. The fundamental reaction of protein synthesis is the formation of a peptide bond between the carboxyl group at the end of a growing polypeptide chain and a free amino group on an incoming amino acid. Consequently, a protein is synthesized stepwise from its N-terminal end to its C-terminal end.
Throughout the entire process the growing carboxyl end of the polypeptide chain remains activated by its covalent attachment to a tRNA molecule a peptidyl-tRNA molecule. This high-energy covalent linkage is disrupted during each addition but is immediately replaced by the identical linkage on the most recently added amino acid Figure The incorporation of an amino acid into a protein.
A polypeptide chain grows by the stepwise addition of amino acids to its C-terminal end. The formation of each peptide bond is energetically favorable because the growing C-terminus has been activated more As we have seen, the synthesis of proteins is guided by information carried by mRNA molecules.
To maintain the correct reading frame and to ensure accuracy about 1 mistake every 10, amino acids , protein synthesis is performed in the ribosome , a complex catalytic machine made from more than 50 different proteins the ribosomal proteins and several RNA molecules, the ribosomal RNAs rRNAs. A typical eucaryotic cell contains millions of ribosomes in its cytoplasm Figure As we have seen, eucaryotic ribosomal subunits are assembled at the nucleolus , by the association of newly transcribed and modified rRNAs with ribosomal proteins, which have been transported into the nucleus after their synthesis in the cytoplasm.
The two ribosomal subunits are then exported to the cytoplasm, where they perform protein synthesis. Ribosomes in the cytoplasm of a eucaryotic cell. This electron micrograph shows a thin section of a small region of cytoplasm. The ribosomes appear as black dots red arrows. Some are free in the cytosol; others are attached to membranes of the endoplasmic more Eucaryotic and procaryotic ribosomes are very similar in design and function.
Both are composed of one large and one small subunit that fit together to form a complete ribosome with a mass of several million daltons Figure The small subunit provides a framework on which the tRNAs can be accurately matched to the codons of the mRNA see Figure , while the large subunit catalyzes the formation of the peptide bonds that link the amino acids together into a polypeptide chain see Figure A comparison of the structures of procaryotic and eucaryotic ribosomes.
Despite the differences in the more When not actively synthesizing proteins, the two subunits of the ribosome are separate. The mRNA is then pulled through the ribosome; as its codons encounter the ribosome's active site , the mRNA nucleotide sequence is translated into an amino acid sequence using the tRNAs as adaptors to add each amino acid in the correct sequence to the end of the growing polypeptide chain.
When a stop codon is encountered, the ribosome releases the finished protein, its two subunits separate again. These subunits can then be used to start the synthesis of another protein on another mRNA molecule. Ribosomes operate with remarkable efficiency: in one second, a single ribosome of a eucaryotic cell adds about 2 amino acids to a polypeptide chain; the ribosomes of bacterial cells operate even faster, at a rate of about 20 amino acids per second.
How does the ribosome choreograph the many coordinated movements required for efficient translation? A tRNA molecule is held tightly at the A- and P-sites only if its anticodon forms base pairs with a complementary codon allowing for wobble on the mRNA molecule that is bound to the ribosome.
This feature of the ribosome maintains the correct reading frame on the mRNA. The RNA-binding sites in the ribosome. A Structure of a bacterial ribosome with more Once protein synthesis has been initiated, each new amino acid is added to the elongating chain in a cycle of reactions containing three major steps. Our description of the chain elongation process begins at a point at which some amino acids have already been linked together and there is a tRNA molecule in the P-site on the ribosome , covalently joined to the end of the growing polypeptide Figure In step 2, the carboxyl end of the polypeptide chain is released from the tRNA at the P-site by breakage of the high-energy bond between the tRNA and its amino acid and joined to the free amino group of the amino acid linked to the tRNA at the A-site, forming a new peptide bond.
This central reaction of protein synthesis is catalyzed by a peptidyl transferase catalytic activity contained in the large ribosomal subunit. This reaction is accompanied by several conformational changes in the ribosome, which shift the two tRNAs into the E- and P-sites of the large subunit.
In step 3, another series of conformational changes moves the mRNA exactly three nucleotides through the ribosome and resets the ribosome so it is ready to receive the next amino acyl tRNA. Step 1 is then repeated with a new incoming aminoacyl tRNA , and so on. Translating an mRNA molecule. Each amino acid added to the growing end of a polypeptide chain is selected by complementary base-pairing between the anticodon on its attached tRNA molecule and the next codon on the mRNA chain.
Because only one of the many more This three-step cycle is repeated each time an amino acid is added to the polypeptide chain, and the chain grows from its amino to its carboxyl end until a stop codon is encountered. The basic cycle of polypeptide elongation shown in outline in Figure has an additional feature that makes translation especially efficient and accurate.
Under some conditions, ribosomes can be made to perform protein synthesis without the aid of the elongation factors and GTP hydrolysis, but this synthesis is very slow, inefficient, and inaccurate. The process is speeded up enormously by coupling conformational changes in the elongation factors to transitions between different conformational states of the ribosome.
Although these conformational changes in the ribosome are not yet understood in detail, some may involve RNA rearrangements similar to those occurring in the RNAs of the spliceosome see Figure Detailed view of the translation cycle. The outline of translation presented in Figure has been supplemented with additional features, including the participation of elongation factors and a mechanism by which translational accuracy is improved.
In addition to helping move translation forward, EF-Tu is thought to increase the accuracy of translation by monitoring the initial interaction between a charged tRNA and a codon see Figure Although the bound elongation factor allows codon- anticodon pairing to occur, it prevents the amino acid from being incorporated into the growing polypeptide chain. The initial codon recognition, however, triggers the elongation factor to hydrolyze its bound GTP to GDP and inorganic phosphate , whereupon the factor dissociates from the ribosome without its tRNA, allowing protein synthesis to proceed.
The elongation factor introduces two short delays between codon-anticodon base pairing and polypeptide chain elongation; these delays selectively permit incorrectly bound tRNAs to exit from the ribosome before the irreversible step of chain elongation occurs. The first delay is the time required for GTP hydrolysis. The rate of GTP hydrolysis by EF-Tu is faster for a correct codon-anticodon pair than for an incorrect pair; hence an incorrectly bound tRNA molecule has a longer window of opportunity to dissociate from the ribosome.
Although this lag is believed to be the same for correctly and incorrectly bound tRNAs, an incorrect tRNA molecule forms a smaller number of codon-anticodon hydrogen bonds than does a correctly matched pair and is therefore more likely to dissociate during this period. These two delays introduced by the elongation factor cause most incorrectly bound tRNA molecules as well as a significant number of correctly bound molecules to leave the ribosome without being used for protein synthesis, and this two-step mechanism is largely responsible for the Recent discoveries indicate that EF-Tu may have an additional role in raising the overall accuracy of translation.
Earlier in this chapter, we discussed the key role of aminoacyl synthetases in accurately matching amino acids to tRNAs. Exactly how this is accomplished is not well-understood, but it may involve the overall binding energy between EF-Tu and the aminoacyl-tRNA. According to this idea, correct matches have a narrowly defined affinity for EF-Tu, and incorrect matches bind either too strongly or too weakly.
EF-Tu thus appears to discriminate, albeit crudely, among many different amino acid-tRNA combinations, selectively allowing only the correct ones to enter the ribosome. The ribosome is a very large and complex structure, composed of two-thirds RNA and one-third protein. The determination, in , of the entire three-dimensional structure of its large and small subunits is a major triumph of modern structural biology.
The structure strongly confirms the earlier evidence that rRNAs—and not proteins—are responsible for the ribosome's overall structure, its ability to position tRNAs on the mRNA , and its catalytic activity in forming covalent peptide bonds.
Thus, for example, the ribosomal RNAs are folded into highly compact, precise three-dimensional structures that form the compact core of the ribosome and thereby determine its overall shape Figure Structure of the rRNAs in the large subunit of a bacterial ribosome, as determined by x-ray crystallography. One of the protein subunits of the ribosome more In marked contrast to the central positions of the rRNA , the ribosomal proteins are generally located on the surface and fill in the gaps and crevices of the folded RNA Figure Some of these proteins contain globular domains on the ribosome surface that send out extended regions of polypeptide chain that penetrate short distances into holes in the RNA core Figure The main role of the ribosomal proteins seems to be to stabilize the RNA core, while permitting the changes in rRNA conformation that are necessary for this RNA to catalyze efficient protein synthesis.
Location of the protein components of the bacterial large ribosomal subunit. For convenience, the protein structures depict only the polypeptide backbones. Structure of the L15 protein in the large subunit of the bacterial ribosome.
The globular domain of the protein lies on the surface of the ribosome and an extended region penetrates deeply into the RNA core of the ribosome. The L15 protein is shown in more Not only are the three binding sites for tRNAs the A-, P-, and E-sites on the ribosome formed primarily by the ribosomal RNAs, but the catalytic site for peptide bond formation is clearly formed by the 23S RNA , with the nearest amino acid located more than 1.
This RNA-based catalytic site for peptidyl transferase is similar in many respects to those found in some proteins; it is a highly structured pocket that precisely orients the two reactants the growing peptide chain and an aminoacyl- tRNA , and it provides a functional group to act as a general acid- base catalyst —in this case apparently, a ring nitrogen of adenine, instead of an amino acid side chain such as histidine Figure The ability of an RNA molecule to act as such a catalyst was initially surprising because RNA was thought to lack an appropriate chemical group that could both accept and donate a proton.
Although the p K of adenine-ring nitrogens is usually around 3. A possible reaction mechanism for the peptidyl transferase activity present in the large ribosomal subunit. The overall reaction is catalyzed by an active site in the 23S rRNA. In the first step of the proposed mechanism, the N3 of the active-site adenine more RNA molecules that possess catalytic activity are known as ribozymes. We saw earlier in this chapter how other ribozymes function in RNA-splicing reactions for example, see Figure In the final section of this chapter, we consider what the recently recognized ability of RNA molecules to function as catalysts for a wide variety of different reactions might mean for the early evolution of living cells.
Here we need only note that there is good reason to suspect that RNA rather than protein molecules served as the first catalysts for living cells. If so, the ribosome , with its RNA core, might be viewed as a relic of an earlier time in life's history—when protein synthesis evolved in cells that were run almost entirely by ribozymes. The initiation and termination of translation occur through variations on the translation elongation cycle described above. The site at which protein synthesis begins on the mRNA is especially crucial, since it sets the reading frame for the whole length of the message.
An error of one nucleotide either way at this stage would cause every subsequent codon in the message to be misread, so that a nonfunctional protein with a garbled sequence of amino acids would result.
The initiation step is also of great importance in another respect, since for most genes it is the last point at which the cell can decide whether the mRNA is to be translated and the protein synthesized; the rate of initiation thus determines the rate at which the protein is synthesized.
We shall see in Chapter 7 that cells use several mechanisms to regulate translation initiation. This initiator tRNA always carries the amino acid methionine in bacteria, a modified form of methionine—formylmethionine—is used so that all newly made proteins have methionine as the first amino acid at their N-terminal end, the end of a protein that is synthesized first.
In RNA, thymine is replaced with uracil in most cases. In DNA, thymine T binds to adenine A via two hydrogen bonds, thus stabilizing the nucleic acid structures.
It is a pyrimidine derivative, with a heterocyclicaromatic ring and two substituents attached an amine group at position 4 and a keto group at position 2.
Thenucleoside of cytosine is cytidine. In Watson-Crick base pairing, it forms three hydrogen bonds with guanine. In DNA, guanine is paired with cytosine. With the formula C5H5N5O, guanine is a derivative of purine, consisting of a fused pyrimidine-imidazole ring system with conjugated double bonds. The guanine nucleoside is called guanosine.
This method will create a degenerate sequence, and where possible, each codon will encode all possible synonymous codons for a particular amino acid. Note that amino acids with 6 synonymous codons Leu, Arg and Ser cannot be represented by a single unambiguous degenerate sequence. The combined YTN will also encode Phe. Check the option "Allow ambiguous codons" if required. Choose whether to use "Preferred Codons" using most frequent synonymous codon only , or "Codons chosen with probabilities based on the frequency distribution".
We recommend you consult the literature for advice on the recommended codon usage frequencies for your expression host. The new "reverse translated" sequence will be annotated with a translated CDS Feature. Double click the feature and rename it if desired. The relative frequencies of synonymous codons observed for the new CDS and the CUT used to generate the new sequence should be roughly the similar.
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