A. Introduction to Translation

Translation is the cellular process that converts the language of nucleic acids contained within the mRNA molecule (A, U, G, C) into the language of amino acids in a synthesized protein (methionine, alanine, histidine, etc.). This translation process relies on a genetic code that converts nucleic acid sequence into amino acid sequence. Note that the genetic code that is translated into protein functions within the mRNA molecule, not in the DNA.

The mRNA is read by the ribosome during translation as a group of three nucleotides (triplet code). Each group of three nucleotides within the mRNA sequence is called a codon.

The mRNA contains several sequence features that play a role in translation (figure 11.1). These sequence features are listed from 5’ to 3’ along the mRNA molecule, as follows:

• 5’ untranslated region (5’-UTR). The 5’-UTR of the mRNA is not translated by the ribosome. Instead, the 5’-UTR allows the ribosome to bind to the mRNA and positions the ribosome to interact properly with the start codon (see below). In bacteria, the 5’-UTR of the mRNA contains a specific nucleotide sequence called the Shine-Dalgarno sequence that serves as the binding site for the ribosome. In eukaryotes, the 5’-UTR contains the 7-methylguanosine (7-mG) cap, which serves as the ribosome binding site, and a specific nucleotide consensus sequence called the Kozak sequence, which then positions the ribosome to recognize the start codon.
• Start codon. The start codon in the mRNA codes for the first amino acid in the synthesized protein. The start codon is usually 5'-AUG-3’ and encodes the amino acid N-formylmethionine (fmet) in bacteria and methionine (met) in eukaryotes.
• Sense codons. After the start codon, the mRNA is read as consecutive three nucleotide-long sense codons. Each sense codon specifies an amino acid in the protein. Note that the start codon and the sense codons constitute the coding region of a gene.
• Stop codon. The stop codon in the mRNA serves as a signal to end translation. The possible stop codons within prokaryotic and eukaryotic mRNAs include 5’-UAG-3’, 5’-UAA-3’, or 5’-UGA-3’. The stop codon does not encode an amino acid within the synthesized polypeptide.
• 3’ untranslated region (3’-UTR). The 3’-UTR of the mRNA, located downstream of the stop codon, plays a role in transcriptional termination. In prokaryotes, the 3’-UTR contains the rut, stem-loop, and the uracil-rich sequences that function in rho (ρ)-dependent and rho (ρ)-independent transcriptional termination (see Part 9). In eukaryotes, the 3’-UTR contains the polyadenylation signal sequences recognized by the CPSF and CstF proteins during transcription termination (see Parts 9 and 10).

The Genetic Code

There are 20 different types of amino acids typically incorporated in proteins; however, there are 64 possible combinations of four nucleotides (A, U, G, and C) in a triplet codon. As a result, there are more codons than amino acid possibilities (see figure 11.2). Thus, the genetic code is degenerate, meaning that more than one codon can encode a particular amino acid type.  For example, the amino acid valine (val) is encoded by four codons (5’-GUU-3’, 5’-GUC-3’, 5’-GUA-3’, and 5’-GUG-3’) that differ only in the 3’-most base (wobble base).  These four valine codons are called synonymous codons.

The genetic code is also unambiguous, meaning that a particular codon sequence only encodes one type of amino acid. For example, the codon 5’-AAA-3’ always encodes the amino acid lysine (lys). Further, the genetic code is commaless, meaning that codons are read by the ribosome consecutively one after another as triplets, with no spacer nucleotides between the codons. The codons are also nonoverlapping, meaning each nucleotide in the coding region of the mRNA is a member of only one codon. Finally, the same genetic code is used in all organisms (in other words, the code is universal).

Directionality of Polypeptides

The polypeptide chains that are synthesized during translation have directionality (polarity); however, since the language of proteins is different than the language of nucleic acids, the labels 5' and 3' do not apply to protein directionality.  Instead, the amino acid encoded by the start codon (closer to the 5’ end of the mRNA) contains a free amino (NH₃⁺) chemical group and thus is said to be the amino or N-terminus of the polypeptide chain. As the polypeptide is synthesized, a peptide bond is formed between the carboxyl group (COO^-) of the growing amino acid chain and the amino group of the incoming amino acid (see figure 11.3).  Peptide bond formation involves a condensation reaction that releases water as a product. The final amino acid added to a polypeptide contains a free carboxyl chemical group and is called the carboxyl terminal or C-terminal end of the polypeptide chain. The C-terminal end of the polypeptide corresponds to the codon immediately before the stop codon (closer to the 3’ end of the mRNA).

tRNAs are an Adaptor Molecule

Transfer RNA molecules (tRNAs) act as adaptor molecules in translation. tRNAs function to:

• Recognize the triplet nucleotide sequence of the codon within the mRNA. This recognition process involves hydrogen bonding between the mRNA codon and an anticodon sequence within the tRNA.
• Carry a specific amino acid. tRNA molecules carry amino acids to the ribosome. The carried amino acid is then incorporated into the growing polypeptide chain. The amino acid carried by the tRNA is specified by the anticodon sequence.

There are many different types of tRNA molecules in a cell. Each type is encoded by a different gene in the genome and has a unique nucleotide sequence. tRNA molecules are named according to the amino acid they carry.  For example, tRNA^val carries the amino acid valine, while tRNA^phe carries the amino acid phenylalanine.

Features of tRNAs

All tRNA molecules have similar structural features (figure 11.4):

• tRNA molecules are small. Each tRNA type (e.g., tRNAval, tRNAphe ) is 75–90 nucleotides in length.
• tRNA molecules are shaped like a cloverleaf. Three stem-loop structures are formed by hydrogen bonding within the tRNA molecule. The anticodon is located within one of these stem-loop structures, called the anticodon loop.
• tRNA molecules contain a 3’ single-stranded region called an acceptor stem. The acceptor stem region is covalently linked to the amino acid.
• tRNA molecules contain variable regions. The tRNA variable regions differ between tRNA types (e.g., tRNA^val, tRNAphe) in the number of nucleotides and the specific nitrogenous base sequence that they contain. Variability in these regions can make the stem loops different sizes.
• tRNA molecules contain unusual nitrogenous bases. Many tRNA molecules have unusual nitrogenous bases, such as inosine (I), methylinosine (mI), and dimethylguanine (m₂G). These unusual nitrogenous bases are created from the four conventional RNA nitrogenous bases (A, G, U, and C) after transcription of the tRNA molecule has occurred.

Charging tRNAs

The process of attaching an amino acid to the 3' end of a tRNA molecule is called charging. Charging tRNAs involves groups of enzymes that are collectively called aminoacyl-tRNA synthetases. Specific features of aminoacyl-tRNA synthetases include:

• There are 20 different groups of aminoacyl-tRNA synthetases per cell, one group for each of the 20 amino acid types.
• Aminoacyl-tRNA synthetases are named for the amino acid that is attached to the tRNA. For example, alanyl-tRNA synthetases attach the amino acid alanine (ala) to a tRNA molecule, while prolyl-tRNA synthetases attach the amino acid proline (pro) to a tRNA molecule.
• Aminoacyl-tRNA synthetases attach the incorrect amino acid to a tRNA rarely. In fact, it is estimated that aminoacyl-tRNA synthetases attach the wrong amino acid one time per 100,000 tRNA molecules charged.

The mechanism used by an aminoacyl-tRNA synthetase to charge a tRNA molecule has the following steps (see figure 11.5):

1. The aminoacyl-tRNA synthetase binds to its preferred amino acid and ATP.
2. The ATP molecule is cleaved, PP_i (pyrophosphate) is released, and the remaining AMP is covalently linked to the amino acid forming an aminoacyladenylic acid intermediate within the enzyme.  When the aminoacyladenylic acid intermediate is produced, the aminoacyl-tRNA synthetase changes its conformation (shape) to produce the active form of the enzyme.
3. The aminoacyl-tRNA synthetase binds to a tRNA molecule that contains an appropriate anticodon sequence. If the anticodon on the incoming tRNA is incorrect, the tRNA is released from the aminoacyl-tRNA synthetase and a new tRNA binds to the enzyme.
4. The aminoacyl-tRNA synthetase attaches the amino acid to the 3’ end of the tRNA.
5. AMP is released.
6. The charged tRNA is released from the aminoacyl-tRNA synthetase.

   

The Wobble Rules

Most synonymous codons (codons that encode the same amino acid) have identical bases at the first two nucleotide positions (the 5’-most base and the middle base of the codon). These first two bases obey the AU/GC base pairing rules when forming hydrogen bonds with the tRNA anticodon. Degeneracy in the genetic code typically occurs at the 3'-most base within the codon. The 3’-most base in the codon does not have to form conventional base pairing interactions with the 5’-most base of the anticodon, leading to “wobble.” The rules that govern codon-anticodon at this wobble position interactions are called the wobble rules (see Table 11.1).  For example, a U base in the wobble position of the codon (3'-most base) can form hydrogen bonds with A, G, or I in the wobble position of the anticodon (5'-most base).  Similarly, a U in the wobble position of the anticodon (5'-most base) can form hydrogen bonds with either A or G in the wobble position of the codon (3'-most base).

A group of tRNA molecules, with slightly different anticodon sequences, which can recognize the same codon are called isoacceptor tRNAs. For example, suppose we have the codon 5’-UUU-3’. A tRNA containing either 3’-AAA-5’, 3’-AAG-5’, or 3’-AAI-5’ anticodons can recognize this codon, according to the wobble rules (see Table 11.1). Note that these isoacceptor tRNAs are charged by the same aminoacyl-tRNA synthetase and therefore, bear the same amino acid. Alternatively, a tRNA with the 3’-GGU-5’ anticodon can recognize two codons: 5’-CCA-3’and 5’-CCG-3’ according to the wobble rules.  Both codons encode proline.

Why would a cell use this wobble phenomenon? Wobble gives the cell tremendous flexibility in codon:anticodon interactions.  A single mRNA codon can be recognized by more than one tRNA anticodon, and a single tRNA anticodon can bind to more than one mRNA codon.  This flexibility allows a cell to save energy in the synthesis of tRNA molecules. Instead of assembling 61 different types of tRNA molecules (one type for each possible sense codon, minus the three stop codons), a cell can get away with synthesizing only 30-40 types of tRNA molecules, with some tRNAs recognizing multiple codon sequences.

Prokaryotic Ribosomes

The ribosome is the central figure in the translation process. The ribosome binds to the mRNA, serves as a site for mRNA codon - tRNA anticodon recognition, breaks the covalent bond between the 3’ end of the tRNA and the amino acid, and synthesizes peptide bonds between amino acids. 

A single bacterial cell is thought to contain 10,000 ribosomes; these ribosomes are called 70S ribosomes. Note that the “S” designation of ribosomes correlates roughly with the overall three-dimensional shape of the ribosome component; the larger the molecule, the larger the “S” value.

The 70S ribosome consists of the following subunits (see figure 11.6):

• Small subunit (30S subunit). The small subunit of the ribosome is composed of several different ribosomal proteins and a 16S ribosomal RNA (rRNA) molecule. The 16S rRNA molecule helps the ribosome bind to the bacterial mRNA molecule (see below).
• Large subunit (50S subunit). The large subunit of the ribosome is composed of many ribosomal proteins, a 5S rRNA, and a 23S rRNA molecule.  The 23S rRNA molecule is the catalytic core of the ribosome; the 23S rRNA is the RNA enzyme (ribozyme) that forms peptide bonds between amino acids during prokaryotic translation (see below).

Eukaryotic Ribosomes

Eukaryotic cells have cytosolic ribosomes called 80S ribosomes. Mitochondrial and chloroplast ribosomes resemble prokaryotic 70S ribosomes.

The 80S ribosomes in the cytosol consist of the following subunits (see figure 11.7):

• Small subunit (40S). The 40S subunit is composed of many proteins and an 18S rRNA molecule.
• Large subunit (60S). The 60S subunit is composed of a collection of proteins, 5S, 5.8S, and 28S rRNA molecules. The 28S rRNA molecule is the RNA enzyme (ribozyme) that forms peptide bonds between amino acids during eukaryotic translation (see below).

Overview of Translation

Translation in prokaryotes and eukaryotes occurs in three stages (see figure 11.8):

1. During initiation, the large and small ribosomal subunits assemble near the start codon within the mRNA that is to be translated and with an initiator tRNA that has an anticodon specific for the start codon. Once all translation components have assembled correctly, the initiator tRNA is located at the P site (see below) within the ribosome.
2. During elongation, the ribosome translocates (moves) along the mRNA in the 5’ to 3’ direction, reading triplet codons within the mRNA, converting the codon nucleic acid sequences into an amino acid chain. During elongation, three tRNA binding sites within the ribosome are used. These tRNA binding sites are the:
   • Aminoacyl site (A site). The A site is the binding site for charged tRNAs that enter the ribosome.
   • Peptidyl site (P site). The polypeptide chain attached to the tRNA molecule is found in the P site of the ribosome. The polypeptide is transferred from the P site to the A site when the peptide bond forms (see below).
   • Exit site (E site). The E site is the exit site for uncharged tRNA molecules.
3. When the ribosome reaches a stop codon, translation terminates. The mRNA and the polypeptide chain are released from the ribosome, and the large and small subunits of the ribosome dissociate from each other.  The ribosome subunits and the mRNA can be recycled to synthesize another copy of the protein.

Shine-Dalgarno Sequence

The 5’-untranslated region (5’-UTR) of prokaryotic mRNAs plays a critical role in translation, as the 5’-UTR contains the ribosome-binding site. Specifically, the 5’-UTR contains a recognition sequence (5’-AGGAGGU-3’) called the Shine-Dalgarno sequence that forms hydrogen bonds with a complementary sequence within the 16S rRNA of the small ribosomal subunit (30S) (see figure 11.9). Hydrogen bond formation between the Shine-Dalgarno sequence and the 16S rRNA promotes ribosome assembly. Further, the Shine-Dalgarno sequence positions the P site of the ribosome properly at the start codon to initiate polypeptide synthesis.

Translation Initiation in Bacteria

Translation initiation in the bacterium E. coli includes the following (see figure 11.10):

1. The mRNA binds to the small ribosomal subunit (30S) via the Shine-Dalgarno sequence. An initiation factor protein called IF3 promotes the formation of hydrogen bonds between the ribosome and the mRNA.
2. An initiator tRNA recognizes the start codon within the P site of the ribosome. The initiator tRNA anticodon forms hydrogen bonds with the mRNA start codon in the P site. In bacteria, this initiator tRNA is covalently attached to a modified form of methionine called N-formylmethionine (fmet). Thus, the N-terminal amino acid in all newly synthesized prokaryotic proteins is fmet. Binding of the tRNA^fmet to the start codon requires another initiation factor protein called IF2 that cleaves GTP to load the tRNA^fmet into the ribosome P site. The complex of the 30S subunit, the mRNA, IF3, and IF2 is called the initiation complex.
3. The 50S subunit of the ribosome is added to the initiation complex. The addition of 50S requires the release of IF2 and IF3.

Translation Initiation in Eukaryotes

Translation initiation is more complex in eukaryotes than in prokaryotes. Key differences between eukaryotic and prokaryotic translation initiation include (see figure 11.11):

• • More initiation factor proteins are involved in eukaryotes. These initiation factor proteins are called eukaryotic initiation factors (eIFs).
  • The initiator tRNA is eukaryotes is tRNA^met. This initiator tRNA is charged with the amino acid methionine (met).
  • There is no Shine-Dalgarno sequence in a eukaryotic mature mRNA. Instead, eukaryotic mature mRNAs contain a 7-methylguanosine (7-mG) cap and a Kozak sequence within the 5’-UTR.

The process of translation initiation in eukaryotes is as follows:

1. The eIF2 protein binds to tRNA^met and guides it to the 40S ribosomal subunit. eIF2 cleaves GTP to deliver tRNA^met to the 40S ribosomal subunit.

2. Assembly of a multi-protein initiation complex on the 7-methylguanosine (7-mG) cap at the 5’ end of the mRNA. This initiation complex includes the following:

• • 40S ribosome subunit
  • A complex composed of eIF2 and tRNA^met
  • An eIF4 protein complex.
  • Cap-binding protein 1 (CBP1). CBP1 is the protein responsible for recognizing the 7-mG structure on eukaryotic mature mRNAs.

3. Identification of the start codon. The 40S ribosomal subunit and associated factors start at the 7-mG cap and move 5’ to 3’ along the mRNA looking for a start codon. This scanning process requires the cleavage of ATP. Not all possible start codons (5’-AUG-3’) are chosen to initiate translation. The 5’-AUG-3’ that is chosen is within the consensus sequence 5’-GCC (A or G)CCAUGG-3’. This consensus sequence is the Kozak sequence. When the tRNA^met forms hydrogen bonds with the start codon, tRNA^met is in the P site of the ribosome.

4. Addition of the 60S ribosomal subunit to form the active 80S ribosome.

Figure 9.11 - Translation initiation in eukaryotes

Translation Elongation

Translation elongation in bacteria involves a series of cycles, one cycle per sense codon in the mRNA. At the beginning of each cycle, the tRNA molecule located within the P site of the ribosome is attached to a polypeptide chain (see figure 11.12). The A and E sites of the ribosome are empty. Each elongation cycle then proceeds as follows:

1. A charged tRNA binds to the A site within the ribosome. The prokaryotic elongation factor protein EF-Tu is responsible for loading a charged tRNA into the A site of the ribosome. This loading of a charged tRNA into the A site requires the cleavage of GTP.
2. The codon and anticodon hydrogen bonds are checked. Of course, the anticodon within the newly delivered tRNA must be complementary to the mRNA codon according to the wobble rules. An incorrect tRNA bound to the A site is recognized by the 16S rRNA. When an incorrect anticodon is encountered, polypeptide synthesis is halted until the mismatched tRNA is released from the ribosome. This editing function of the 16S rRNA molecule allows for high fidelity in protein synthesis; it is estimated that only one mistake is made per 10,000 incorporated amino acids, or about one time in every 20 proteins synthesized by the ribosome.
3. The formation of a peptide bond. The polypeptide chain bound to the tRNA in the P site is transferred to the tRNA in the A site via the formation of a new peptide bond. This enzyme reaction is catalyzed by the 23S rRNA component of the 50S ribosomeal subunit; the 23S rRNA is an example of a ribozyme (RNA enzyme). The 23S rRNA is also called peptidyl transferase.
4. Ribosome translocation. The ribosome translocates (moves) in the 5’ to 3’ direction one codon along the mRNA. The tRNA (with the attached polypeptide chain) that was in the A site moves to the P site. The uncharged tRNA that was in the P site moves to the E site and exits the ribosome. A bacterial elongation factor protein called EF-G cleaves GTP to translocate the ribosome to the next sense codon in the mRNA.
5. Steps 1–4 are repeated for each sense codon in the mRNA. The rate of protein synthesis is 15–18 cycles (i.e., 15-18 amino acids) per second in prokaryotes.

Translation elongation works similarly in both prokaryotes and eukaryotes; however below are some minor differences to keep in mind for eukaryotic translation:

• The 28S rRNA component of the 60S ribosomal subunit functions as the peptidyl transferase to synthesize the peptide bonds between amino acids. The 28S rRNA is another example of a ribozyme (RNA enzyme).
• Eukaryotes have eukaryotic elongation factors (eEFs) that function very similarly to EF-Tu and EF-G from bacteria. The eukaryotic eEF1 protein functions similarly to EF-Tu in bacteria, delivering charged tRNA molecules to the A site of the ribosome. The eukaryotic eEF2 protein functions similarly to EF-G in bacteria, translocating the ribosome one codon towards the 3’ end of the mRNA after peptide bond formation has taken place. Both eEF1 and eEF2 cleave GTP molecules.

Translation Termination

Translation termination in bacteria occurs as follows (see figure 11.13):

1. As the ribosome approaches the 3’ end of the mRNA, a stop codon (5’-UGA-3’, 5’-UAG-3’, or 5’-UAA-3’) moves into the A site of the ribosome.
2. The stop codon is recognized by a release factor protein. The release factor protein mimics the three-dimensional structures of tRNA molecules. In bacteria, the release factor 1 (RF1) protein recognizes the stop codons 5’-UAA-3’ and 5’-UAG-3’. The release factor 2 (RF2) protein recognizes 5’-UAA-3’ and 5’-UGA-3’.
3. The release factor protein cleaves the covalent bond between the tRNA and the polypeptide chain in the ribosome P site. As a result, the tRNA and the polypeptide chain are released from the ribosome. This step requires the release factor protein to cleave GTP.
4. The ribosomal subunits, mRNA, and release factor protein dissociate, terminating translation.  The ribosome subunits and the mRNA can be recycled to begin translation again.

Translation termination works essentially the same way in eukaryotes. One minor diference is the use of a single eukaryotic release factor (eRF) protein that recognizes all three possible stop codons. eRF cleaves GTP to terminate translation.

Polysomes and Coupled Transcription and Translation

In the electron microscope, multiple ribosomes (polyribosomes or polysomes) are often observed attached to a single mRNA transcript (see figure 11.14). Polysomes have been observed attached to both prokaryotic mRNA and eukaryotic mature mRNA transcripts.

Since prokaryotic cells lack nuclei, transcription and translation are coupled, meaning that translation can begin before transcription is completed. In eukaryotes, transcription and translation are uncoupled. Transcription and RNA modifications occur within the nucleus; translation occurs later, in the cytoplasm.

Review Questions

Fill in the blanks:

1. The ______________ portion of a eukaryotic mature mRNA contains the polyadenylation signal sequences.
2. The genetic code is said to be __________________, which means that each codon only specifies one type of amino acid. (For example, 5’-UUU-3’ always encodes phenylalanine)
3. The cloverleaf structure of the tRNA is such that the ____________________________ is a single-stranded loop that forms hydrogen bonds with the mRNA codon, and the ___________________________ is the single-stranded region where an amino acid is attached via a covalent linkage.
4. ____________________________ is an enzyme that links the amino acid to a tRNA molecule.
5. The 30S and 50S ribosomal subunits in a prokaryotic cell combine to form a final size of _________ while in eukaryotes the _________ and __________ ribosomal subunits combine to form a final size of ______________.
6. The initiator tRNA binds to the __________ site of the ribosome, whereas the remaining charged tRNAs bind to the ___________ site of the ribosome.
7. An E. coli protein called _______________ delivers charged tRNAs to the ribosome during translation elongation.
8. The ribosome moves along the mRNA in the _______to _______ direction.
9. _______________ is a eukaryotic translation factor protein that delivers charged tRNAs to the ribosome during elongation.
10. ___________ is a eukaryotic translation factor that helps the ribosome move one codon in the 3’ direction along the mRNA.
11. The _________________ protein in bacteria mimics the structure of a tRNA and recognizes 5’-UGA-3’ in the A site of the ribosome.
12. An E. coli protein called __________ delivers tRNA^fmet to the ribosome during translation.