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The Genetic Code
& Translation
At the heart of the central dogma is the concept that information in the form of the four-letter alphabet (A, G, C and T) of the genetic material is translated into the 20-letter (amino acids) alphabet of proteins. As we have seen, the intermediary between the genetic material, DNA, and the translation machinery, the ribosome , is the messenger RNA or mRNA. The mRNA is copied from the DNA in a process called transcription (Chapter
- and is then decoded on the ribosome in a process called translation , where it directs the ordered polymerization of amino acids into polypeptide chains. Here we focus on the nature and logic of the genetic code , the RNA adaptors that decipher the genetic code, the workings of the molecular machine that translates mRNA into protein and does so with high accuracy, and the chemistry of the formation of the peptide bond.
The four-letter alphabet of the genetic material is read in units of three How many bases are required to specify an amino acid? Because nucleic acids have only four bases and proteins are composed of twenty amino acids, the coding unit or codon for each amino acid must consist of more than one base. Even two bases would not be enough. If codons consisted of two bases, then only 16 (or 4^2 ) different codons would be possible, and there would insufficient codons to specify 20 amino acids. However, if codons are composed of three bases, then 64 (or 4^3 ) codons are possible, more than enough for the amino acid alphabet. Therefore, the minimum number of bases needed to specify 20 different amino acids is three. Indeed, the genetic code is a triplet code.
- describe the principal features of the genetic code.
- explain how tRNAs mediate information transfer and do so accurately.
- describe the peptide bond cycle and how it achieves accuracy.
- describe the ribosome cycle and how open reading frames are set.
After this chapter, you should be able to
To understand how mRNA is translated into protein.
Objectives
Goal
Of the 64 possible codons in the genetic code, 61 specify amino acids, indicating that many amino acids are encoded by more than one codon (Figure 1). Thus, the code is degenerate in the sense that some amino acids are specified by more than one synonymous codon. At one extreme, leucine, serine, and arginine are each specified by six synonymous codons. At the other extreme, methionine and tryptophan have unique codons (AUG and UGG, respectively). The methionine codon AUG has an additional function as a start codon : it signals the beginning of a coding sequence in the mRNA. The remaining three (of the 64) triplets—UAA, UGA, and UAG—do not specify any amino acids. Instead, these triplets are stop codons that signal the end of the coding sequence for a messenger RNA. Thus, coding sequences have two kinds of punctuation marks: an AUG at the beginning that marks the start and one (or more successive) stop codons at the end. We will return to start and stop codons near the end of the chapter, including the question of how AUG can serve both as a methionine codon internal to a coding sequence and as the start signal at the beginning. The entire genetic code, which is sometimes called the “Rosetta Stone of Life” (because it deciphers codons), is shown in its entirety in Figure 1. Notice that the left-hand vertical column indicates the first (5’) position in a codon, the horizontal bar across the top indicates the second position, and the right-hand vertical column indicates the third (3’) position. Start and stop codons are highlighted in green and red, respectively. Finally, we return to the 5’-to-3’ directionality of polynucleotides. Codons have a 5’-to-3’ orientation with respect to the directionality of the RNA
Figure 1 Each codon corresponds
to a particular amino acid
Each codon is written from 5’ to 3’. The start codon (AUG) is shown in green. Stop codons (UAA, UAG, and UGA) are shown in red.
U C^ A^ G
U
C
A
G
First Position (5’)
Second position
Third position (3’)
UUU
UUC
UUA
UUG
Phe
Leu
UCU
UCC
UCA
UCG
Ser
Pro
Thr
Ala Gly
Leu
UAU
UAC
UAA
UAG
Tyr
Stop
UGU
UGC
UGA
UGG
U C A G U C A G U C A G U C A G
Cys
Stop Trp
CUU CUC CUA CUG
CCU
CCC
CCA
CCG
CAU
CAC
CAA
CAG
His
Gln
CGU
CGC
CGA
CGG
AUU
AUC
AUA
AUG
Ile
Met
ACU
ACC
ACA
ACG
AAU
AAC
AAA
AAG
Asn
Lys
AGU
AGC
AGA
AGG
Ser
Arg
Arg
GUU
GUC
GUA
GUG
Val
GCU
GCC
GCA
GCG
GAU
GAC
GAA
GAG
Asp
Glu
GGU
GGC
GGA
GGG
Aminoacyl-tRNA synthetases catalyze tRNA charging What is the nature of the linkage between an amino acid and a tRNA, and how is it created? Amino acids are joined to tRNAs via an acyl linkage between the carboxyl group of a cognate amino acid and the 3’ hydroxyl at the 3’ end of the tRNA molecule, creating an aminoacyl-tRNA (Figure 4). tRNAs bearing an aminoacyl linkage are said to be charged. Charging is catalyzed by an enzyme called aminoacyl-tRNA synthetase (Figure 5). Each amino acid has its own aminoacyl-tRNA synthetase (meaning there are 20 synthetases), which recognizes a particular amino acid and all of the cognate tRNAs for that amino acid.
amino acid attachment point
5’ 3’
anti-codon
3’
5’
anti-codon
amino acid attachment point
(A) (B)
Figure 3 tRNAs exhibit secondary and tertiary structure
(A) Shown is a cartoon representing the two-dimensional, cloverleaf-like secondary structure of a generic tRNA. Each circle represents an individual nucleotide, with the nucleotides of the anti-codon shown in blue. Black lines indicate base pairing within the strand. (B) Shown is the X-ray crystal structure of a tRNA molecule. The tRNA folds into an L-shaped structure, with the anti-codon at one end and the 3’ hydroxyl, which is where the amino acid becomes attached, at the other end. All tRNAs have this overall folded structure, even though their specific nucleotide sequences vary. Shown is a tRNA for phenylalanine; as such, it is known as tRNAPhe.
Figure 4 tRNA charging creates a high-energy acyl linkage between the tRNA and its cognate amino acid
The particular tRNA represented has a 3’-AAC-5’ anti-codon, which is complementary to the leucine codon 5’-UUG-3’. Consequently, this tRNA (“tRNALeu”) is being charged with leucine.
OH
AAC
OH
O
NH (^2)
O
H 2 N (^) O
AAC
ATP
AMP, PP (^) i
high-energy acyl linkage
anti-codon (3’ AAC 5’)
3’ hydroxyl
tRNA Leu
Charging is energetically unfavorable; it has a ΔG°rxn that is positive. Therefore, energy must be expended in order to create the aminoacyl linkages. Charging takes place in a two-step process in which the formation of the aminoacyl linkage is coupled to the hydrolysis of ATP (Figure 6). The first step is a transesterification reaction in which the adenosine monophosphate (AMP) moiety of ATP is transferred to the carboxyl group of the amino acid, producing aminoacyl-AMP. This transfer occurs by nucleophilic attack by the oxygen atom from the carboxyl group on the α-phosphate of the ATP, resulting in the liberation of pyrophosphate. As we saw in the context of DNA polymerization, the liberation of pyrophosphate helps drive the reaction forward in that it is coupled to the favorable hydrolysis of pyrophosphate by pyrophosphatase. In the second step, the aminoacyl group is transferred from the aminoacyl-AMP intermediate to the tRNA, generating aminoacyl-tRNA and releasing AMP. This second step is favorable because the reactant aminoacyl-AMP is of higher free energy than the product aminoacyl-tRNA. As we will come to, the aminoacyl-tRNA is the direct substrate for peptide bond formation by the ribosome. Peptide bond formation between a free amino group of one amino acid and the free carboxyl group of another amino acid is energetically unfavorable and hence does not occur spontaneously. However, the aminoacyl linkage in the charged tRNA reactant is at a higher free energy state than the peptide-bond product. Thus, the energy released by the expenditure of an ATP molecule is transferred first to the formation of aminoacyl-AMP, then to the formation of aminoacyl-tRNA and ultimately to the formation of a peptide bond by the ribosome.
tRNA
anti-codon
catalytic binding pocket
editing pocket
aminoacyl-tRNA synthetase
Figure 5 Aminoacyl-tRNA
synthetases catalyze tRNA charging
Shown is the X-ray crystal structure of the isoleucyl-tRNA synthetase bound to a tRNAIle. This particular enzyme contains both a catalytic site with a binding pocket for isoleucine and an editing site with a pocket that distinguishes isoleucine from the closely related amino acid valine.
side chains display a negative charge could be excluded from the binding pocket of the catalytic site for an amino acid whose side chain carries a positive charge. Therefore, in many cases, differences in size and chemistry among amino acids suffice for effective discrimination. In some cases, however, the synthetase must distinguish between amino acids that are so similar in size and chemical properties that effective discrimination based solely on selective binding is not possible. Consider, for example, the challenge of the synthetase for isoleucine in discriminating against valine. Isoleucine differs from valine only by the presence of an extra methyl group in its side chain (Figure 7). Hence, valine is likely to fit in the catalytic-site binding pocket for isoleucine. Even though the catalytic site favors isoleucine over valine, the difference in ΔG°rxn between isoleucine binding and valine binding is only 2.5 kcal/mol. This means (recall from Chapter 9 that 2.7 kcal/mol roughly corresponds to an equilibrium constant of 100) that the isoleucine aminoacyl-tRNA synthetase would bind valine and incorporate the wrong amino acid about once every 100 times, which is a high error rate. How then does the synthetase for isoleucine achieve high accuracy? It does so using a proofreading mechanism analogous to the editing pocket we encountered in Chapter 9 for DNA polymerase. Isoleucyl-tRNA synthetase has an editing pocket that enables it to discriminate against valine in either of the two steps of charging. Recall that isoleucine is successively transferred to AMP and then to the tRNA. If mis-acylation takes place with valine instead of isoleucine, then the valine moiety, whether attached to AMP or to tRNA, is able to slip into an editing pocket that is only large enough to allow valine with its smaller side chain to enter (for simplicity, Figure 8 only shows editing at the second, tRNA aminoacylation step). Once in the editing pocket, the aminoacyl linkage is hydrolyzed, allowing the synthetase to begin a fresh charging cycle. Editing improves the accuracy of charging to less than one mis-charging event in 10,000 cycles of aminoacylation.
The ribosome is a molecular machine Arguably, the ribosome is the most extraordinary of all the molecular machines that mediate the processes of living systems. This will become self- evident as we proceed. The ribosome is composed of two subunits known as the large subunit and small subunit. The total mass of the ribosome is about 3,000 kilodaltons (1 kilodalton = 1,000 atomic mass units), about six times the size (~ 500 kilodaltons) of RNA polymerase. Each subunit is a complex of proteins and RNA molecules. The RNA molecules, which are known as ribosomal RNAs or rRNAs, are non-protein-coding RNAs, which
H 3 N
O
O
CH (^3)
CH (^3)
H 3 N
O
O
H 3 C CH (^3)
isoleucine valine
Figure 7 The aminoacyl-tRNA
synthetase for isoleucine must discriminate against valine, which differs from isoleucine by only a methyl group
as we will see are intimately involved in decoding mRNA and in protein synthesis. In bacteria, the small subunit consists of a 1,540-nucleotide-long rRNA and 21 proteins, whereas the large subunit contains two rRNAs, one of 120 nucleotides and the other of 2,900 nucleotides, and 31 proteins. The ribosomes of eukaryotes are somewhat larger and more complex. Ribosomes contain three binding sites for tRNAs that are important landmarks in the process of translation (Figure 9). These are the A-site , the P-site , and the E-site , which each span across the large and small subunits. The A-site is the entry site for aminoacyl-tRNAs. The P-site is the site at which the peptidyl-tRNA, the growing polypeptide chain attached to tRNA, is located. Finally, the E-site is the exit site from which tRNA that has handed off its cargo leaves the ribosome. As we shall see, all three sites participate dynamically in translation as one charged tRNA after another delivers amino acids for incorporation into the growing polypeptide chain.
Peptide bond formation takes place in three steps In overview, translation commences at the beginning of a protein-coding sequence (which we will soon refer to as an open reading frame) where the start codon is located and terminates at the end of the coding sequence, which is marked by one or more stop codons. Near the end of this chapter we will return to the molecular events that trigger the start and stop of translation. For now, we focus on the ribosome in steady state as it migrates
Ile
Val
Isoleucine added (correct)
Valine added (incorrect)
Ile
Val
catalytic binding pocket
editing pocket tRNA
aminoacyl-tRNA synthetase
Ile
Val
Isoleucine is too large to enter editing pocket
Isoleucine remains attached to the tRNA
Valine is small enough to enter editing pocket
The editing pocket removes valine from the tRNA
Figure 8 Isoleucyl-tRNA synthetase has an editing pocket that discriminates against valine
the free amino group of the glycine and the carbonyl carbon that is connected to the tRNA by an acyl linkage. As a consequence of peptide bond formation, the acyl linkage between the peptide and the tRNAArg^ in the P-site is broken and the peptide chain that is now one residue longer is transferred to the tRNAGly^ in the A-site. Thus, peptide bond formation involves the transfer of the growing chain from the tRNA in the P-site to the tRNA in the A-site. In step 3 , the ribosome translocates one codon unit (three nucleotides) along the mRNA in the 5’-to-3’ direction, thereby shifting the peptidyl- tRNAGly^ into the P-site, shifting the tRNAArg^ now freed of its cargo into the E-site and leaving the A-site vacant. Once in the E-site, the deacylated tRNAArg^ dissociates from the ribosome, leaving the E-site vacant. The now-empty A-site is ready to accept another charged tRNA in the next cycle of peptide bond formation. Notice in our example that the peptide chain is growing in an NH 2 -to-COOH-terminal direction and that the mRNA is being translated in a 5’-to-3’ direction, in keeping with the directional rules we introduced in Chapter 8 and the 5’-to-3’ orientation of codons.
CCU
G
AUG ACG CGA GGA UUG GGA
GCU
R
M T
GCU
R
M T
AUG ACG CGA GGA UUG GGA
CCU
G
GCU
R
M T
AUG ACG CGA GGA UUG GGA
CCU
G GCU
R
M T
AUG ACG CGA GGA UUG GGA
CCU
G
AAC
L
R
M T
AUG ACG CGA GGA UUG GGA
CCU
G
R
M T
AUG ACG CGA GGA UUG GGA
CCU
G
AAC
L
R
M T
AUG ACG CGA GGA UUG GGA
CCU AAC
L
G R
M T
AUG ACG CGA GGA UUG GGA GG U
CCU AAC
L
G
mRNA small subunit
large subunit P-site
aminoacyl-tRNA
5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’
5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’
E E E
E E E
A A A
A A A
Growing peptide (N-terminus)
Step 1 The aminoacyl-tRNA that matches the codon binds to the empty A-site.
Step 2 A peptide bond forms between the amino acid in the A-site and the growing peptide chain in the P-site. The ribosome translocates by three nucleotides, and the growing peptide chain is transferred onto the tRNA that was previously in the A-site.
Step 3 The small subunit shifts posi- tions and the tRNA in the E-site is eject- ed. This resets the ribosome for anoth- er translation cycle. Next cycle
Figure 10 Peptide bond formation during elongation is a cyclical process that sequentially adds individual
amino acids to the COOH-terminus of the growing polypeptide Shown here are two cycles of elongation. In the first cycle (top row), a glycine residue is added to the COOH-terminus of the growing polypeptide chain. In the second cycle (bottom row), a leucine residue is added. Notice that in translocation (step 3) the large subunit moves ahead first and is then joined by the small subunit.
Entry of charged tRNA into the A-site is mediated by Elongation Factor Tu with the expenditure of a molecule of GTP In step 1 of the peptide bond formation cycle, a charged tRNA enters the A-site of the ribosome. Entry into the A-site does not, however, occur with free molecules of aminoacyl-tRNA. Rather, charged tRNAs are escorted into the A-site in a complex with a protein called Elongation Factor Tu or EF-Tu bound to a molecule of the guanine nucleotide GTP (EF-Tu·GTP) (Figure 11). Accuracy in protein synthesis demands that the correct charged tRNA enter the A-site as dictated by the codon exposed on the mRNA in the A-site. To ensure that the correct charged tRNA has entered the A-site, EF-Tu·GTP releases its cargo of charged tRNA if, and only if, correct pairing takes place between the anti-codon and the codon. If not, the complex of charged tRNA and EF-Tu·GTP simply diffuses away (Figure 12). If so, then the GTP is hydrolyzed before the complex diffuses away. Whether GTP hydrolysis takes place before the complex diffuses away is governed by the length of time that the charged tRNA remains in the A-site, which is in turn determined by whether there is a correct codon/anti-codon match. Hydrolysis causes a change in the conformation of the EF-Tu that causes the complex to dissociate, allowing free, charged tRNA to remain in the A-site. Thus, energy in the form of GTP is expended to ensure that only the correct charged tRNA is deposited into the A-site. Indeed, without this energy-dependent certification it would not be possible for the ribosome to achieve a high level of accuracy in protein synthesis.
GTP
aminoacyl-tRNA
EF-Tu
Figure 11 The GTP-bound
conformation of EF-Tu binds tightly to aminoacyl-tRNAs
Shown is the X-ray crystal structure of the GTP-bound conformation of EF-Tu bound to an aminoacyl-tRNA. Hydrolysis of GTP to GDP alters the shape of EF-Tu, causing its release from the aminoacyl-tRNA.
Peptide bond formation is mediated by nucleophilic attack of the free amino group of the incoming amino acid and the carboxyl carbon of the growing polypeptide chain As we have seen, peptide bond formation takes place in step 2 of the cycle. What is the chemical mechanism by which the bond is formed, and what enzyme catalyzes the reaction? In peptide bond formation, a lone pair of electrons on the nitrogen of the free amino group of the amino acid in the A-site attacks the carboxyl carbon joining the peptide chain to the tRNA in the P-site (i.e., the carbonyl carbon at the C-terminus of the growing peptide chain) (Figure 13). Thus, in peptide bond formation nucleophilic attack replaces the acyl linkage between the carbonyl carbon and the 3’ hydroxyl of the tRNA with a peptide bond between the carbonyl carbon and the amino nitrogen. Recall that the aminoacyl linkage to the tRNA is a high- energy bond that was created with the expenditure of a molecule of ATP during tRNA charging. Thus, energy spent in the form of ATP hydrolysis during tRNA charging is harnessed on the ribosome to drive peptide bond formation without the input of any additional source of energy. The enzyme that catalyzes peptide bond formation is known as peptidyl transferase. The catalytic center for the peptidyl transferase sits in the large subunit of the ribosome. In one of the most amazing discoveries in molecular biology, structural and biochemical experiments have shown that the peptidyl transferase catalytic center is largely, if not entirely, composed of the 2,900-nucleotide rRNA of the large subunit. Thus, an RNA molecule catalyzes peptide bond formation. The peptidyl transferase is therefore an example of an RNA enzyme or ribozyme , a topic to which we return in Chapter 13. Thus, at the heart of the ribosome is an RNA molecule that generates peptide bonds, the most fundamental feature of proteins.
O H O
O R (^3)
NH O
R (^2) HN
H 3 N O
R (^1)
O
N O R (^4)
H
H
aminoacyl-tRNA empty tRNA
peptidyl-tRNA
O
O R (^4)
NH O
R (^3) HN
NH O R (^2)
O
H 3 N
R (^1)
new peptidyl-tRNA
peptide bond formation
Figure 13 During peptide bond formation the growing peptide is transferred from the peptidyl-tRNA to the
aminoacyl-tRNA
Shown is the arrow-pushing mechanism for the peptide bond-forming reaction that takes place during translation. The free amino group on the amino acid attached to the aminoacyl-tRNA acts as a nucleophile that attacks the carbonyl carbon at the COOH-terminus of the polypeptide attached to the peptidyl-tRNA. The acyl linkage that connects the growing polypeptide to the peptidyl-tRNA is broken. As a consequence, a new peptidyl-tRNA is generated from the previous aminoacyl-tRNA. This reaction is also known as peptidyl transfer, as the entire peptide is transferred from the peptidyl-tRNA to the aminoacyl-tRNA.
Movement of the ribosome along the mRNA following peptide bond formation is driven by Elongation Factor G with the hydrolysis of a molecule of GTP Finally, after the peptide bond has formed, the ribosome must translocate along the mRNA by three nucleotides so that the next codon can enter the A-site and the next cycle of translation can commence. How does this movement take place, and what is the source of energy that drives
Box 1
Antibiotics kill bacteria by binding to and blocking the functions of various molecular machines in the cell. One of the most common targets of antibiotics is the ribosome. Examples of antibiotics that target the ribosome include erythromycin, kanamycin, streptomycin, and tetracycline. Figure 14 shows the X-ray crystal structure of erythromycin (green) bound to a bacterial ribosome. This particular antibiotic binds close to the site where the peptidyl transferase reaction takes place, and in doing so, inhibits protein synthesis. Importantly, erythromycin is able to discriminate between bacterial and human ribosomes; otherwise, it would be toxic.
Many antibiotics target the ribosome
erythromycin
aminoacyl-tRNA
peptidyl-tRNA
peptidyl transfer
location
Figure 14 Erythromycin binds to the ribosome and disrupts translation
The large ribosomal subunit is shown in cyan; rRNA is omitted for clarity, and only proteins are shown. The small ribosomal subunit is shown in green. The aminoacyl-tRNA is shown in magenta and the peptidyl-tRNA is shown in orange. The location of the peptidyl transferase reaction is indicated, with the two amino acid substrates shown in the box.
to the cap (Chapter 10) at the 5’ end of the mRNA. The small subunit then slides down the mRNA in a 5’-to-3’ direction while scanning for a 5’- AUG-3’. The first 5’-AUG-3’ it encounters serves as the start codon. Once a start codon is encountered, the large subunit is recruited to the mRNA- bound small subunit by initiation factors to create the initiation complex. In eukaryotes, the start codon simply specifies methionine rather than a modified form of methionine. Finally, we come to the dissociation of the ribosome when it reaches the end of a protein-coding sequence. The end of the coding sequence is marked by a stop codon (5’-UAA-3’, 5’-UGA-3’, or 5’-UAG-3’) or sometimes more than one stop codon in succession. Stop codons are not recognized by tRNAs. Instead, stop codons are recognized by release factors , which catalyze the release of the completed polypeptide chain from the peptidyl-tRNA and the dissociation and release of the ribosome from the mRNA (Figure 16). Thus, the cycle of association and dissociation repeats with each round of translation, with the small and large subunits assembling into an initiation complex at the start of a protein-coding sequence and dissociating back into free subunits at the end of a coding sequence. Lastly, it is important to note that the start site of translation is not the same as the start site of transcription. Indeed, the start codon is preceded by untranslated sequences that extend upstream to the 5’ end of the mRNA, which corresponds to the transcription start site (position +1 in the transcription unit). As we have seen, this upstream region contains the ribosome binding site in bacteria and untranslated sequences downstream of the 5’ cap that the small subunit scans in eukaryotes. Likewise, the stop codon does not correspond to the 3’ end of an mRNA. Rather, the 3’ end of the mRNA extends past the coding sequence and contains untranslated sequences.
The start codon sets the reading frame for protein-coding sequences As we have seen, the formation of the initiation complex at the beginning of a protein-coding sequence is a complicated process involving multiple protein factors and recognition cues in the mRNA. Why is it so complicated?
E P A
R
M T
AUG ACG CGA GGA UGA GGA
CCU
G
5’ 3’
E A
R
M T
AUG ACG CGA GGA UGA GGA
CCU
G
5’ 3’
E AUG ACG CGA GGA UGA GGA
CCU 5’ 3’
E 5’AUG ACG CGA GGA UGA GGA 3’
CCU
H 2 O
R
M T
G
N-term
free C-term
release factor
N-term large subunit
small subunit
Release factor binds to stop codon
Peptide chain is hydrolyzed from peptidyl-tRNA
Ribosome dissociates from mRNA
1 2 3
Figure 16 Release factors recognize stop codons and terminate translation
Why do bacteria and eukaryotes go to such lengths to ensure that protein synthesis is initiated with precision at the start codon? The answer is that the start codon not only marks the beginning of a protein-coding sequence but also sets the reading frame in which all the successive triplets will be translated. Without a start codon, the mRNA is simply a string of nucleotides that could in principle be translated in any of three possible reading frames. Just which frame is the correct one is set by the start codon. That is, each successive and immediately adjacent triplet after the start codon is in the same reading frame as the start codon and represents the correct coding sequence. If, for example, the position of the start codon in the RNA were shifted by one or two nucleotides, then the downstream coding sequence would be completely altered. Consider, for example, the three sequences shown in Figure 17. All three are the same except for the positions of the 5’-AUG-3’ start codons, which set three different reading frames. In this hypothetical example, three completely different amino acid sequences are translated from the same RNA sequence depending on the frame of the start codon. These considerations explain why initiation must take place with single- nucleotide precision; otherwise, the ribosome would generate the wrong sequence of amino acids. Note also that the reading frame determines whether the ribosome will encounter a stop codon. Stop codons are only recognized as such if they are in the same reading frame as the amino-acid-specifying codons that precede them in a protein-coding sequence. For these reasons, protein- coding sequences are often referred to as open reading frames , that is, a stretch of base triplets that lacks, or extends up to, a stop codon in the same frame. Thus, exons in the pre-mRNAs of eukaryotes are open reading frames. Removal of an intron between two exons merges the exons into a single, longer open reading frame. This explains why, as stated in Chapter 10, removal of introns from pre-mRNAs by splicing must also take place with single-nucleotide precision. If not, then the ribosome could or would translate the mRNA in an incorrect frame, resulting in a completely different sequence of amino acids. Finally, the concept of an open reading frame also explains why mutations that insert or delete a single base pair in a coding sequence ( frame shift mutations ) have profound effects on the function of a gene as compared to mutations that simply replace one base pair with another. Insertions and deletions change the reading frame, whereas replacement of one base pair with another only alters a single codon.
Met Thr Thr Thr Thr Thr Thr Thr
Met Asp Asp Asp Asp Asp Asp Asp
Met Arg Arg Arg Arg Arg Arg Arg
5’ ... AUGACGACGACGACGACGACGACG... 3’
AUGGACGACGACGACGACGACGACG
AUGCGACGACGACGACGACGACGACG
Reading frame 1
Reading frame 2
Reading frame 3
Figure 17 mRNA sequences can
be decoded in three reading frames
Shown is the repeating sequence 5’-… ACGACGACGACG…-3’ translated in three different reading frames, which are set by the placement of the start codon.
terminates at stop codons, which are recognized by release factors that release the completed polypeptide, completing the ribosome cycle. Thus, protein synthesis involves two cycles: cycles of peptide bond formation during elongation that are embedded within cycles of ribosome assembly and disassembly on the mRNA.
In addition to marking the beginning of a protein-coding sequence, the start codon sets the reading frame for downstream codons. The protein- coding sequence is therefore also known as an open reading frame.
