Cover1 - Chromosome Structure2 - Chromosome Compaction3 - Chromosome Variation5 - Nucleic Acid Structure6 - DNA Replication7 - Mutations and DNA Repair8 - Polymerase Chain Reaction (PCR)9 - Transcription10 - RNA Modifications11 - Translation12 - Gene Cloning13 - The lac Operon14 - Gene Regulation in Eukaryotes15 - Epigenetics16 - Genome Editing

11 - Translation

A. Introduction to Translation

Translation is the cellular process that translates the language of nucleic acids (A, U, G, C) contained within the mRNA molecule into the language of amino acids (met, ala, his, etc.) in a synthesized protein. This translation process relies on a code that converts nucleic acid sequence into amino acid sequence (genetic code). 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:

9.1_Features_of_Prokaryotic_and_Eukaryotic_mRNA.jpg
Figure 11.1 - Prokaryotic and eukaryotic mRNA features. --- Image created by SL

Key Questions

  • Identify one important sequence feature found in the 5’-UTR in bacteria.
  • Identify two important sequence features found within the 5’-UTR of eukaryotes.
  • What amino acid is encoded by 5’-AUG-3’ in bacteria?
  • What amino acid is encoded by 5’-AUG-3’ in eukaryotes?
  • What is the function of the coding region?
  • What is the function of the stop codon?
  • Does the stop codon encode an amino acid?
  • What is the function of the 3’-UTR?

The Genetic Code

There are 20 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 codon 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).

9.2_The_Genetic_Code.jpg
Figure 11.2 The genetic code. This representation of the genetic code is read from the center of the circle (the 5’-most base in the codon) to the periphery of the circle (the 3’- most base in the codon). Aminoacids Table was created by Mouagip and is used under CC0

Key Questions

  • How would you use the words degenerate, unambiguous, commaless, nonoverlapping, and universal to describe the genetic code?

Directionality of Polypeptides

The polypeptide chains that are synthesized during translation have directionality. The amino acid encoded by the start codon (closer to the 5’ end of the mRNA) contains a free amino (NH3+) group and thus is said to be the amino or N-terminus of the polypeptide.

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 group and is called the C-terminal or carboxyl terminal end. This end of the polypeptide corresponds to a codon closer to the 3’ end of the mRNA.

9.3_Peptide_Bond_Formation.jpg
Figure 11.3 Peptide bond formation --- Image Peptide Bond Formation was created by Yassine Mrabet and used under CC0

Key Questions

  • How do the N- and C-terminal ends of a protein compare to the 5' and 3' ends of an mRNA molecule?

The Adaptor Hypothesis

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

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

Key Questions

  • What are the two functions of a tRNA molecule?

Features of tRNAs

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

9.4_tRNA_Structure.jpg
Figure 11.4 tRNA Structure --- Image used from OpenStax (access for free at https://books.byui.edu/-vuzA)

Key Questions

  • Where is the anticodon in a tRNA molecule?
  • Where is the amino acid attached to a tRNA molecule?

Charging tRNAs

The process of attaching an amino acid to a tRNA molecule is called charging. Amino acids are attached to the 3’ ends of tRNAs using enzymes called aminoacyl-tRNA synthetases. Specific features of aminoacyl-tRNA synthetases include:

The catalytic mechanism of an aminoacyl-tRNA synthetase is as follows (see figure 11.5):

  1. The aminoacyl-tRNA synthetase binds to its preferred amino acid and ATP.
  2. ATP is cleaved, pyrophosphate is released, and the AMP is covalently linked to the amino acid forming an aminoacyladenylic acid intermediate.  When the aminoacyladenylic acid intermediate is produced, the aminoacyl-tRNA synthetase changes its conformation 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.
    EDI_11.5_Charging_a_tRNA_Molecule-01_1.jpg
    Figure 11.5 Charging a tRNA Molecule --- Image by Dr. Frank Boumphrey used under license CC BY-SA 3.0

Key Questions

  • Which molecule provides the energy for the tRNA charging reaction?
  • Explain the major events that occur during the tRNA charging reaction.

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). These first two bases obey the AU/GC base pairing rules in terms of codon-anticodon hydrogen bonding. Degeneracy in the genetic code typically occurs at the third base within the codon. The 3’-most nucleotide in the codon does not have to form conventional base pairing interactions with the 5’-most nucleotide of the anticodon, leading to “wobble.” The rules that govern codon-anticodon interactions are called the wobble rules (see Table 11.1).

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. Note that these isoacceptor tRNAs are charged by the same aminoacyl-tRNA synthetase and 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’.  Both codons encode proline.

Why would a cell use this wobble phenomenon? Wobble gives the cell tremendous flexibility in codon:anticodon interactions.  A single codon can be recognized by more than one tRNA anticodon, and a single tRNA can bind to more than one 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), a cell can get away with synthesizing only 30-40 types of tRNA molecules, with several tRNA types recognizing multiple codon sequences.

9.1_Table_Wobble_Rules.jpg

Table 11.1 Wobble Rules Table

Key Questions

  • What is meant by wobble?
  • What are isoacceptor tRNAs?
  • What is the advantage of wobble?

Prokaryotic Ribosomes

The ribosome is a central figure in the translation process. The ribosome binds to the mRNA, serves as a site for codon-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 prokaryotic cell is thought to contain 10,000 bacterial ribosomes (70S ribosomes). Note that the “S” designation of ribosomes is named after the scientist Theodor Svedberg and describes the behavior of the ribosome during ultracentrifugation. The “S” designation 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 (see figure 11.6):

EDI_11.6_Prokaryotic_Ribosomes-01_1.jpg
Figure 11.6 Prokaryotic Ribosomes --- Prokaryotic cell (Left) used from OpenStax (access for free at https://books.byui.edu/-vuzA).Image created by SL

Eukaryotic Ribosomes

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

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

EDI_11.7_Eukaryotic_ribosomes-01_1.jpg
Figure 11.7 Eukaryotic ribosomes --- Image created by SL

Key Questions

  • Compare and contrast the structural features of prokaryotic vs. eukaryotic ribosomes.
  • What is the name of the ribozyme within the 70S ribosome?
  • What is the name of the ribozyme within the 80S ribosome?

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 with 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. The ribosome moves or translocates along the mRNA in the 5’ to 3’ direction, reading the nucleic acid sequence of the mRNA as triplet codons, converting the nucleic acid sequence into a polypeptide chain. During elongation, three tRNA binding sites within the assembled 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 ends. The mRNA and the polypeptide chain are released from the ribosome, and the large and small subunits of the ribosome dissociate from each other.
EDI_11.8_Overview_of_Translation-01_1.jpg
Figure 11.8 Overview of Translation --- image used from OpenStax (access for free at https://books.byui.edu/-vuzA), modified by SL

Key Questions

  • What major events are occurring during the initiation, elongation, and termination stages of translation?
  • What are the functions of the A site, the P site, and the E site?

Shine-Dalgarno Sequence

The 5’-untranslated region (5’-UTR) of prokaryotic mRNAs plays a critical role in translation; 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.

EDI_11.9_the_Shine-Dalgarno_Sequence-01_1.jpg
Figure 11.9 The Shine-Dalgarno Sequence --- image created by Alejandro Porto and modified by SL. Used under license CC BY-SA 3.0

Key Questions

  • What is the function of the Shine-Dalgarno sequence?

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 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 tRNAfmet to the start codon requires another initiation factor protein called IF2 that cleaves GTP. 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.
9.10_Translation_Initiation_in_Bacteria.jpg
Figure 11.10 Translation initiation in bacteria --- Image created by JET

Key Questions

  • How do IF2 and IF3 function in translation initiation in bacteria?
  • Which nucleotide provides the energy for translation initiation in bacteria?

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):

The process of translation initiation in eukaryotes is as follows:

1. The eIF2 protein binds to tRNAmet and guides it to the 40S ribosomal subunit. eIF2 cleaves GTP to accomplish this function.

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

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 tRNAmet interacts with the start codon, tRNAmet is in the P site of the ribosome.

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

9.11_Eukaryotic_Translation_Initiation.jpg

Figure 9.11 - Translation initiation in eukaryotes

Key Questions

  • How do eIF2 and CBP1 function in translation initiation in eukaryotes?
  • What is the function of the Kozak sequence?
  • Which two nucleotides provide the energy for 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, a growing polypeptide chain attached to a tRNA molecule is located within the P site of the ribosome (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. A prokaryotic elongation factor protein called 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. This editing function of the 16S rRNA 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 polypeptides 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 enzymatic reaction is catalyzed by the 23S rRNA component of the 50S subunit. As a result, the 23S rRNA is often called peptidyl transferase. The 23S rRNA is an example of a ribozyme (catalytic RNA molecule).
  4. Ribosome translocation. The ribosome moves (translocates) 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 14 are repeated for each sense codon in the mRNA. The rate of protein synthesis is 15–18 amino acids per second in prokaryotes.

Translation elongation works similarly in both prokaryotes and eukaryotes. Below are some differences to keep in mind for eukaryotic translation:

EDI_11.12_Translation_elongation_in_bacteria-01_1.jpg
Figure 11.12 Translation elongation in bacteria --- mage used from OpenStax (access for free at https://books.byui.edu/-vuzA), modified by SL

Key Questions

  • Describe the events involved in one translation elongation cycle.
  • Explain the steps where GTP is used as an energy source.
  • What is the eukaryotic equivalent of 23S rRNA, EF-Tu, and EF-G?

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’) becomes located in the A site of the ribosome.
  2. The stop codon is recognized by a translation release factor protein. A 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. The tRNA and the polypeptide chain are released from the ribosome. This step consumes GTP.
  4. The ribosomal subunits, mRNA, and release factor protein dissociate, terminating translation.

Translation termination works essentially the same way in eukaryotes. In terms of the termination step in eukaryotes, a single release factor protein called eukaryotic release factor (eRF) recognizes all three possible stop codons. eRF consumes GTP to terminate translation.

EDI_11.13_Translation_termination-01_1.jpg
Figure 11.13 Translation termination  --- Image used from OpenStax (access for free at https://books.byui.edu/-vuzA), modified by SL

Key Questions

  • What is the difference between RF1 and RF2?
  • What nucleotide provides the energy to terminate translation?
  • Discuss what happens to each of the individual molecules after translation is terminated. Are some of them recycled? If so, which ones?

Polysomes and Coupled Transcription and Translation

In the electron microscope, many ribosomes (polyribosomes or polysomes) can be observed, attached to a single mRNA transcript (see figure 11.14). Polysomes are visualized attached to both prokaryotic mRNA and eukaryotic mature mRNA transcripts.

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

9.14_Polyribosomes.jpg
Figure 11.14 Polyribosomes and coupled transcription/translation in bacteria --- Image used from OpenStax (access for free at https://books.byui.edu/-vuzA)

Key Questions

  • Why is it advantageous to have multiple ribosomes working simultaneously?
  • Why is prokaryotic transcription and translation coupled?
  • Why is eukaryotic transcription and translation uncoupled?

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 charges the amino acid to a tRNA.
  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 assembled ribosome contains three tRNA binding sites. The site in which a tRNA carrying a single amino acid resides is called the _____ site.
  7. The initiator tRNA binds to the __________ site of the ribosome, whereas the remaining charged tRNAs bind to the ___________ site of the ribosome.
  8. An E. coli protein called _______________ delivers charged tRNAs to the ribosome during translation elongation.
  9. The ribosome moves along the mRNA in the _______to _______ direction.
  10. _______________ is a eukaryotic translation factor protein that delivers charged tRNAs to the ribosome during elongation.
  11. ___________ is a eukaryotic translation factor that helps the ribosome move one codon in the 3’ direction along the mRNA.
  12. The _________________ protein in bacteria mimics the structure of a tRNA and recognizes 5’-UGA-3’ in the A site of the ribosome.
  13. An E. coli protein called __________ delivers tRNAfmet to the ribosome during translation.

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