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In this article we will discuss about the introduction and mechanisms of translation in eukaryotes.
Introduction to Translation in Eukaryotes:
The process of protein synthesis from amino acid sequences specified by the sequence of codons in messenger RNA is called translation. Translation is the first stage of protein biosynthesis.
The main points about translation in eukaryotes are given below:
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1. Site:
Translation occurs in the cytoplasm where the ribosomes are located. Ribosomes are made of a small and large subunit which surrounds the mRNA. In eukaryotic translation 80S ribosomes with 40S and 60S subunits are used. The mRNA is synthesized from DNA only. In eukaryotes, there is single initiation and termination site.
2. Template:
This uses an mRNA sequence as a template to guide the synthesis of a chain of amino acids that form a protein. Many types of transcribed RNA, such as transfer RNA, ribosomal RNA, and small nuclear RNA are not necessarily translated into an amino acid sequence.
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3. Requirements:
The translation process requires mRNA, rRNA, ribosomes, 20 kinds of amino acids and their specific tRNAs.
4. Factors Involved:
In eukaryotes, several factors are used in chain initiation such as eIF2, eIF3, eIF4A, eIF4E, eIF4F and elF 4G. Two factors [EF-1 and EF-2] are used in chain elongation. There is a single release factor RF for recognition of three termination codons [UAA, UAG and UGA].
5. Enzymes Involved:
In eukaryotes, two types of enzymes are used in translation. Aminoacyl tRNA synthetase (an enzyme) catalyzes the bonding between specific tRNAs and the amino acids. The enzyme peptidyl transferase connect A site and P site by forming a peptide bond [the nitrogen carbon bond] during elongation phase.
6. Codons Involved:
In the process of translation two types of codons, viz., start codorl and stop codons are involved. The codon, AUG. initiates the process of translation and one of three stop codons i.e. UAA, UAG, or UGA is used for chain termination. In eukaryotes and archaea, the amino acid encoded by the start codon is methionine.
7. Starting Amino Acid:
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In eukaryotes, starting amino acid is methionine. Moreover, there is no overlapping of transcription and translation.
Mechanism of Translation in Eukaryotes:
The mechanism of translation in eukaryotes is similar to that of prokaryotes in several aspects.
Translation process consists of three phases or stages, viz:
(i) Initiation,
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(ii) Elongation and
(iii) Termination.
These are discussed as follows:
1. Initiation:
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The process of initiation of translation in eukaryotes is of two types, viz:
(i) Cap-dependent initiation, and
(ii) Cap-independent initiation.
i. Cap-Dependent Initiation:
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Initiation of translation usually involves the interaction of certain key proteins with a special tag bound to the 5′-end of an mRNA molecule, the 5′ cap. The protein factors bind the small ribosomal subunit (also referred to as the 40S subunit), and these initiation factors hold the mRNA in place.
The eukaryotic Initiation Factor 3 (eIF3) is associated with the small ribosomal subunit, and plays a role in keeping the large ribosomal subunit from prematurely binding.
The factor eIF3 also interacts with the eIF4F complex which consists of three other initiation factors [eIF4A, eIF4E and eIF4G]. The factor eIF4G is a protein which directly associates with both eIF3 and the other two components.
The eIF4E is the cap-binding protein. It is the rate-limiting step of capdependent initiation, and is often cleaved from the complex by some viral proteases to limit the cell’s ability to translate its own transcripts.
The eIF4A is an ATP-dependent RNA helicase, which aids the ribosome in resolving certain secondary structures formed by the mRNA transcript. There is another protein associated with the eIF4F complex called the Poly-A Binding Protein (PABP), which binds the poly-A tail of most eukaryotic mRNA molecules. This protein is considered to play a role in circularization of the mRNA during translation.
This pre-initiation complex (43S subunit, or the 40S and mRNA) along with protein factors move along the mRNA chain towards its 3′-end. It scans for the ‘start’ codon (typically AUG) on the mRNA. The start codon indicates the site where the mRNA will begin coding for the protein. In eukaryotes and archaea, the amino acid encoded by the start codon is methionine.
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The initiator tRNA charged with Met forms pan of the ribosomal complex and thus all proteins start with this amino acid. The Met-charged initiator tRNA is brought to the P-site of the small ribosomal subunit by eukaryotic Initiation Factor 2 (eIF2). It hydrolyzes GTP, and signals for the dissociation of several factors from the small ribosomal subunit which results in the association of the large subunit (or the 60S subunit).
The complete ribosome (80S) then commences translation elongation, during which the sequence between the ‘start’ and ‘stop’ codons is translated from mRNA into an amino acid sequence. In this way a protein is synthesized.
ii. The Cap-Independent Initiation:
This is lesser known method of translation in eukaryotes. This method of translation has been recently discovered. It has been found to be important in conditions that require the translation of specific mRNAs. It works despite cellular stress or the inability to translate most mRNAs. Examples of such type of translation are factors responding to apoptosis and stress-induced responses.
The best studied example of the cap-independent mode of translation initiation in eukaryotes is the Internal Ribosome Entry Site (IRES) approach. The main difference between cap-independent translation and cap-dependent translation is that the former does not require the ribosome to start scanning from the 5′ end of the mRNA cap until the start codon.
The ribosome can be trafficked to the start site by ITAFs (IRES trans-acting factors) bypassing the need to scan from the 5′ end of the un-translated region of the mRNA.
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2. Elongation:
Elongation is dependent on eukaryotic elongation factors At the end of the initiation step, the mRNA is positioned so that the next codon can be translated during the elongation stage of protein synthesis.
The initiator tRNA occupies the P site in the ribosome; and the A site is ready to receive an aminoacyl-tRNA. During chain elongation, each additional amino acid is added to the nascent polypeptide chain in a three-step micro-cycle.
The steps in this micro-cycle are:
(i) Positioning the correct aminoacyl-tRNA in the A site of the ribosome;
(ii) Forming the peptide bond and
(iii) Shifting the mRNA by one codon relative to the ribosome.
The translation machinery works relatively slowly compared to the enzyme systems that catalyze DNA replication. Proteins are synthesised at a rate of only 18 amino acid residues per second, whereas bacterial replisomes synthesize DNA at a rate of 1,000 nucleotides per second.
This difference in rate reflects, in part, the difference between polymerizing four types of nucleotides to make nucleic acids and polymerizing 20 types of amino acids to make proteins. Testing and rejecting incorrect aminoacyl- tRNA molecules takes time and slows protein synthesis.
The rate of transcription in prokaryotes is approximately 55 nucleotides per second, which corresponds to about 18 codons per second, or the same rate at which the mRNA is translated.
In bacteria, translation initiation occurs as soon as the 5′ end of an mRNA is synthesized, and translation and transcription are coupled. This tight coupling is not possible in eukaryotes because transcription and translation are carried out in separate compartments of the cell (the nucleus and cytoplasm).
Eukaryotic mRNA precursors must be processed in the nucleus [e.g., capping, polyadenylation, splicing) before they are exported to the cytoplasm for translation.
3. Termination:
This is the last phase of translation. Termination occurs when one of the three termination codons moves into the A site. These codons are not recognized by any tRNAs.
Termination of elongation is dependent on eukaryotic release factors In eukaryotes, there is only one release factor that is eRF, which recognizes all three stop codons [in place of RF1, RF2, or RF3 factors in prokaryotes]. However, the overall process of termination is similar to that of prokaryotes.
Prokaryotic Versus-Eukaryotic Translation:
The basic steps involved in protein synthesis are similar in both prokaryotes and eukaryotes. However, protein synthesis differs in several aspects in these two groups (Table 24.1).
Central Dogma:
The central dogma of molecular biology was first enunciated by Francis Crick in 1958 and re-stated in a Nature paper published in 1970.
The Central Dogma of Genetics is that:
DNA is transcribed to RNA which is translated to protein. Protein is never back-translated to RNA or DNA; and except for retroviruses, DNA is never created from RNA. Furthermore, DNA is never directly translated to protein. The dogma is DNA to RNA to protein. In other words, ‘once information gets into protein, it can’t flow back to nucleic acid.’