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3.4Translation of Genes

3.4.1

Synthesis of Aminoacyl-tRNA

tRNA

Most tRNA types have intramolecular double strands, and schematically consist of three loops and one stem (Fig. 3-11A). A fourth variable loop may or may not exist. An anticodon loop has an anticodon sequence that is paired with a codon (code) on mRNA. The 3’ end has the CCA-3’ sequence common to all tRNA, and amino acids are bound to the sequence. The actual three-dimensional structure is compact, as shown in Figure. 3-11B.

Aminoacyl-tRNA Synthetase

The correct pairing of each amino acid with tRNA that has a corresponding anticodon is critically important - in the same way that dictionaries are important in translation - in accurately translating a base sequence to an amino acid sequence. Aminoacyl-tRNA synthetase binds an amino acid to the correct tRNA to form aminoacyl-tRNA. This enzyme reacts by recognizing amino acids and, at the same time, the tRNA structure.

Formylmethionyl-tRNA

When a protein is synthesized, the first amino acid code is for methionine. However, in prokaryotes, the first amino acid is formylmethionine - formylated*6 methionine. The two types of tRNA that bind to methionine are tRNAMet and tRNAfMet, and following the binding of methionine to tRNAfMet (i.e., the formation of Met-tRNAfMet) formylation occurs to create fMet-tRNAfMet. Met-tRNAMet is not formylated, and is used for the methionines inside proteins. In eukaryotes, formylation does not occur, and Met-tRNAMet may be used for the first or subsequent methionines.

*6
Formylation: The binding of the formyl group (-CHO). In the formylation of methionine, one of the hydrogen atoms of the amino group of methionine is replaced with the formyl group.

Fig. 3-11. Structure of tRNA

(A) Cloverleaf model
(B) Tertiary Structure

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Structure of E. Coli Ribosomes

Ribosomes are schematically drawn in the shape of a flattened snowman consisting of large and small subunits, but their actual shape is complex (Column Fig. 3-1). The size of ribosome RNA in eukaryotes is larger, with a higher number of proteins.

Column Fig. 3-1. Ribosomes of E. coli

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3.4.2

Ribosomes

What is a ribosome?

Ribosomes are the places where protein synthesis occurs. In both prokaryotes and eukaryotes, a ribosome is a pairing of one large subunit and one small subunit (Column Fig. 3-1). Each subunit is a complex consisting of rRNA and many types of protein. Since RNA is larger and the number of protein types is higher in eukaryotes, prokaryotes have 70S ribosomes and eukaryotes have 80S ribosomes. Ribosomes contain many types of protein, but quantitatively they are rich in RNA, with proteins covering only parts of the surface (two thirds are RNA and one third is proteins). In particular, the space between the two subunits - the place where protein synthesis occurs - consists almost entirely of RNA.
Ribosomes bind to mRNA and interact with aminoacyl-tRNA, activating both of them, and perform enzymatic reactions such as cleaving ester bonds between tRNA and peptide chains and forming peptide bonds between peptides and amino acids. These important functions of ribosomes are carried out by rRNA. Ribosomes are considered to be ribozymes consisting of RNA with enzymatic activity.

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Initiation of Translation

The initiation of translation is in fact a complex reaction (Column Fig. 3-2). First, initiation factors (IFs) dissociate the large and small subunits, and the small subunit is bound to Met-tRNA (fMet-tRNA in prokaryotes) with mRNA and IFs attached. The large subunit then binds to it, forming a complex consisting of a ribosome, mRNA and Met-tRNA. This is known as an initiation complex.

Column Fig. 3-2. Formation of initiation complexes in eukaryotic organisms

An “e” as the first letter in the names of initiation factors represents eukaryotes.

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Elongation of Peptide Chains

The elongation reaction of peptide chains is also complex (Column Fig. 3-3). At the onset of this reaction, Met-tRNA is situated at the P site, and aminoacyl-tRNA bound with an elongation factor (EF) binds to the A site. The ester bonds between the first amino acid (methionine) and tRNA are then cut, and the methionine and an amino acid at the A site form peptide bonds. Reactions then occur from the action of other elongation factors, the vacated tRNA is transferred to the E site, and the peptidyl-tRNA*7 is transferred to the P site. At the same time, the ribosome moves three bases on the mRNA. When the vacated tRNA is detached from the E site, the ribosome returns to its original state. In this way, the elongation reaction is continuously repeated. During this process, two GTP molecules are hydrolyzed for each amino acid added. The first amino acid of proteins synthesized is always methionine, which is synthesized from the side of the free amino group (N-terminal) to the carboxyl group (C-terminal).

*7
Peptidyl-tRNA: tRNA that is bound with peptides and formed on ribosomes in the process of producing proteins

Column Fig. 3-3. Elongation Reaction of Peptide Chains

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3.4.3

Structure of mRNA

Fig. 3-12. Structure of mRNA

In both prokaryotes and eukaryotes, the functional structure of mRNA schematically consists of a 5’ non-coding region, a coding region and a 3’ non-coding region arranged side by side (Fig. 3-12). AUG, the translation initiation codon, is located at the first part of the coding region. The 5’ non-coding region in prokaryotes often contains a sequence complementary to 16S rRNA, to which ribosomes bind. In eukaryotes, no such sequence exists; instead, there are proteins that bind to the cap structure at the 5’ end, forming an appropriate bond between mRNA and ribosomes. The 3’ non-coding region of mRNA in eukaryotes has a sequence related to the degradation rate of mRNA. The coding region between the two non-coding regions encodes an amino acid sequence of a protein.

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Termination of Translation

The termination reaction occurs when the next codon of mRNA is the termination codon (Column Fig. 3-4). A releasing factor (RF) involved in this reaction enters the A site corresponding to the termination codon of mRNA. A peptidyl-tRNA moves to the P site, and a peptide and tRNA are hydrolyzed by the enzymatic action of rRNA, which releases the protein and subsequently tRNA and mRNA, thereby terminating translation.

Column Fig. 3-4. Termination reaction of translation

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3.4.4

Protein Synthesis

During protein synthesis, a reaction continuously occurs in which three bases of mRNA (a codon) and three bases of aminoacyl-tRNA (an anticodon) form pairs. In this reaction, amino acids are arranged by tRNA in accordance with the order of the mRNA codes (Fig. 3-13), the amino acids and tRNA are dissociated and the amino acids are linked. Through this process, amino acids are connected following the order of the mRNA codes. The series of reactions that occur on ribosomes is known as translation.
One strand of mRNA has multiple ribosomes attached that concurrently synthesize proteins, and longer strands of mRNA have more ribosomes attached to them. Clusters of ribosomes bound to mRNA are called polysomes (or polyribosomes), and cells that actively synthesize proteins have many polysomes. The rate at which amino acids are linked is thought to be around 20 per second in prokaryotes. Assuming that the average molecular weight of amino acids is 114, one minute is needed to synthesize a protein with a molecular weight of approximately 135,000. This means that most proteins, with their molecular weights being around 50,000, are generated within 30 seconds. The rate is slower in eukaryotes at around two amino acids per second.
See the Column for more details on the rather complex processes of initiation, elongation and termination of translation.

Fig. 3-13. Schamatic diagram of protein synthesis

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3.4.5

Coordination between Transcription and Translation

Fig. 3-14. Coupling of transcription and translation in prokaryotes

In prokaryotes, protein synthesis is initiated while mRNA is still being synthesized. In genes, the reactions shown in Figure. 3-14 occur; before the completion of an mRNA molecule, the synthesis of other mRNA molecules is initiated in series, and protein synthesis using the mRNA is initiated. In some cases, mRNA degradation is initiated from the 5’ end while mRNA synthesis is still occurring at the 3’ end. The half-life of mRNA in E. coli is therefore generally very short, lasting only several minutes.
In eukaryotes, on the other hand, the pre-mRNA synthesized undergoes processing, and the complete mRNA is transferred from the nucleus into the cytoplasm. The mRNA is not necessarily used for protein synthesis immediately in the cytoplasm. Thus, eukaryotes differ greatly from prokaryotes in that transcription and translation are spatially and temporally separated (see Fig. 4-3 in Chapter 4). In eukaryotes, the half-life of mRNA varies from only several minutes to very long periods.

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21st Amino Acid

The amino acid known as cysteine has a sulfur atom. Another amino acid called selenocystein, which has selenium instead of sulfur, is found in various enzymes and proteins (albeit in minute amounts), and plays a number of important roles. Selenocystein is not produced by cysteine being modified with selenium after its integration into proteins. Selenocysteinyl-tRNAsec is formed by a special converting enzyme, and is used to synthesize proteins on ribosomes. tRNAsec recognizes UGA on mRNA - normally a termination codon. A special sequence is located immediately downstream of UGA, and selenocysteinyl-tRNAsec is used by the action of special translation factors to generate proteins that contain selenocysteine.

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