Prokaryotic Translation (Protein Synthesis)

RNA and DNA are two types of nucleic acids that store and transmit genetic information in living cells. They are composed of nucleotides, which are the building blocks of these molecules. Nucleotides have three parts: a nitrogenous base, a pentose sugar and a phosphate group.

The nitrogenous bases in RNA are adenine (A), guanine (G), cytosine (C) and uracil (U), while in DNA they are adenine (A), guanine (G), cytosine (C) and thymine (T). The pentose sugar in RNA is ribose, while in DNA it is deoxyribose. The phosphate group links the sugar of one nucleotide to the sugar of the next nucleotide, forming a backbone for the nucleic acid strand.

RNA and DNA differ in their structure and function. RNA is usually single-stranded, meaning it has only one nucleotide chain, while DNA is usually double-stranded, meaning it has two complementary nucleotide chains that form a double helix. RNA can fold into various shapes and forms, while DNA has a more rigid and stable structure.

RNA and DNA also have different roles in protein synthesis. RNA is involved in both transcription and translation, while DNA is only involved in transcription. Transcription is the process of copying a segment of DNA into a complementary strand of RNA, called messenger RNA (mRNA). Translation is the process of decoding the mRNA into a sequence of amino acids, which are the building blocks of proteins.

RNA has several types that perform different functions in protein synthesis. mRNA carries the genetic code from DNA to the ribosomes, where translation occurs. Transfer RNA (tRNA) brings the specific amino acids to the ribosomes according to the codons on the mRNA. Ribosomal RNA (rRNA) forms part of the ribosomes and catalyzes the formation of peptide bonds between amino acids. Other types of RNA, such as small nuclear RNA (snRNA) and microRNA (miRNA), are involved in regulating gene expression and processing mRNA.

DNA is the main repository of genetic information in most living organisms. It contains the instructions for making all the proteins that are essential for life. DNA is organized into units called genes, which code for specific proteins or traits. Each gene has a promoter region, which signals where transcription should start, and a terminator region, which signals where transcription should end. Between these regions, there are exons, which are the coding sequences that will be translated into proteins, and introns, which are the non-coding sequences that will be removed from mRNA.

DNA is replicated before cell division to ensure that each daughter cell receives an identical copy of genetic material. The process of DNA replication involves unwinding the double helix, breaking the hydrogen bonds between the complementary bases, and using each strand as a template for synthesizing a new complementary strand. The enzyme DNA polymerase catalyzes this reaction by adding nucleotides to the 3` end of the growing strand. The result is two identical copies of DNA, each consisting of one original strand and one new strand.

In summary, RNA and DNA are two types of nucleic acids that store and transmit genetic information in living cells. They differ in their structure and function, but they work together to enable protein synthesis. RNA is involved in both transcription and translation, while DNA is only involved in transcription. RNA has several types that perform different functions in protein synthesis, while DNA is organized into units called genes that code for specific proteins or traits. DNA is replicated before cell division to ensure that each daughter cell receives an identical copy of genetic material.

Ribosomes are the molecular machines that carry out protein synthesis in all living cells. They are composed of two subunits, a large one (50S) and a small one (30S), that are made of ribosomal RNA (rRNA) and proteins. The subunits are named according to their sedimentation coefficients in a centrifuge.

The ribosomes can be found either freely floating in the cytosol or attached to the endoplasmic reticulum (ER), a membrane-bound organelle. The ribosomes that are bound to the ER are called rough ER and are involved in synthesizing proteins that are destined for secretion or insertion into membranes. The ribosomes that are free in the cytosol are responsible for making proteins that function within the cell.

The main function of the ribosomes is to read the genetic information encoded in the messenger RNA (mRNA) and translate it into a sequence of amino acids, the building blocks of proteins. The mRNA is a copy of a gene that is transcribed from the DNA in the nucleus. The mRNA carries the information in the form of codons, which are triplets of nucleotides that specify which amino acid to add next.

The ribosomes use transfer RNA (tRNA) molecules as adapters to match the codons on the mRNA with the corresponding amino acids. Each tRNA has an anticodon, which is a triplet of nucleotides that is complementary to a codon on the mRNA, and an amino acid attachment site, where a specific amino acid is covalently linked by an enzyme called aminoacyl-tRNA synthetase.

The ribosomes have three binding sites for tRNAs: the A site, the P site and the E site. The A site is where the incoming aminoacyl-tRNA binds to the codon on the mRNA. The P site is where the tRNA carrying the growing polypeptide chain is located. The E site is where the tRNA that has donated its amino acid exits the ribosome.

The process of protein synthesis consists of three stages: initiation, elongation and termination. During initiation, the small ribosomal subunit binds to the mRNA near its 5` end and scans for the start codon (AUG), which signals the beginning of translation. A special initiator tRNA carrying N-formylmethionine (fMet) binds to this codon and occupies the P site. Then, the large ribosomal subunit joins and forms a complete ribosome.

During elongation, the ribosome moves along the mRNA from 5` to 3` direction and adds one amino acid at a time to the polypeptide chain. This is done by bringing in a new aminoacyl-tRNA to the A site, forming a peptide bond between its amino acid and the one on the P site, and shifting both tRNAs to the next sites (P to E and A to P). This cycle repeats until a stop codon (UAA, UAG or UGA) is reached on the mRNA.

During termination, no tRNA recognizes the stop codon and instead a protein called release factor binds to it and triggers the release of the polypeptide chain from the P site. The ribosome also dissociates into its subunits and releases the mRNA and tRNAs.

The newly synthesized polypeptide may undergo further modifications such as folding, cleavage or addition of other molecules before becoming a functional protein.

Each prokaryotic ribosome has two subunits: a small one (30S) and a large one (50S). The subunits are composed of protein and ribosomal RNA (rRNA). The subunits come together to form a functional ribosome when they bind to a messenger RNA (mRNA) molecule. The ribosome reads the mRNA sequence from the 5` to 3` direction and synthesizes the corresponding protein from amino acids.

The ribosome has three binding sites for transfer RNA (tRNA) molecules, which carry amino acids to the ribosome. These sites are called the aminoacyl-tRNA binding site (or A site), the peptidyl-tRNA binding site (or P site), and the exit site (or E site).

• The A site is where the incoming aminoacyl-tRNA binds to the mRNA codon. The codon is a triplet of nucleotides that specifies an amino acid. The aminoacyl-tRNA has an anticodon that is complementary to the codon. For example, if the codon is AUG, which codes for methionine, the anticodon of the aminoacyl-tRNA is UAC.
• The P site is where the tRNA linked to the growing polypeptide chain is bound. The polypeptide chain is a sequence of amino acids that forms the protein. The tRNA in the P site has a peptide bond between its amino acid and the last amino acid of the polypeptide chain. For example, if the polypeptide chain ends with alanine, the tRNA in the P site has alanine attached to it.
• The E site is where the tRNA that has donated its amino acid to the polypeptide chain leaves the ribosome. The tRNA in the E site is deacylated, meaning that it has no amino acid attached to it. For example, if the tRNA in the P site transfers its alanine to the polypeptide chain, it moves to the E site and becomes deacylated.

All three sites (A, P and E) are formed by the rRNA molecules in the ribosome. The rRNA also catalyzes the formation of peptide bonds between amino acids and facilitates the movement of tRNAs and mRNA along the ribosome. The rRNA is therefore essential for protein synthesis.

The process of protein synthesis involves three stages: initiation, elongation and termination. During initiation, the ribosome binds to the mRNA and recognizes the start codon (AUG) that marks the beginning of protein synthesis. During elongation, the ribosome adds one amino acid at a time to the polypeptide chain by moving along the mRNA and matching each codon with its corresponding aminoacyl-tRNA. During termination, the ribosome encounters a stop codon (UAA, UAG or UGA) that signals the end of protein synthesis and releases the finished polypeptide.

In this article, we will focus on how each stage of protein synthesis works in prokaryotes. Prokaryotes are organisms that lack a nucleus and other membrane-bound organelles. Examples of prokaryotes are bacteria and archaea. Prokaryotic translation differs from eukaryotic translation in some aspects, such as the location, speed and regulation of protein synthesis.

Translation is the process of converting the information encoded in mRNA into a sequence of amino acids that form a protein. Translation occurs in three main stages: initiation, elongation and termination.

Initiation

Initiation is the first stage of translation, where the mRNA, the ribosome and the initiator tRNA come together to form a complex. The initiator tRNA carries the amino acid methionine (or N-formylmethionine in prokaryotes) and recognizes the start codon AUG on the mRNA.

In prokaryotes, initiation involves the following steps:

• The small ribosomal subunit (30S) binds to three initiation factors (IF-1, IF-2 and IF-3) that prevent its premature association with the large subunit (50S).
• The small subunit scans the mRNA for a purine-rich sequence called the Shine-Dalgarno sequence, which is complementary to a region of the 16S rRNA in the small subunit. This sequence helps align the start codon AUG with the P site of the ribosome.
• The initiator tRNA, charged with N-formylmethionine and bound to IF-2 and GTP, binds to the start codon in the P site via codon-anticodon base pairing.
• The large ribosomal subunit (50S) joins the complex, displacing IF-1 and IF-2 and hydrolyzing GTP. This forms a 70S initiation complex that is ready for elongation.

Elongation

Elongation is the second stage of translation, where the polypeptide chain grows by adding one amino acid at a time. Elongation involves three steps: aminoacyl-tRNA binding, peptide bond formation and translocation.

Aminoacyl-tRNA binding

In this step, an aminoacyl-tRNA that matches the next codon on the mRNA binds to the A site of the ribosome via codon-anticodon interaction. This requires elongation factor EF-Tu and GTP, which form a complex with the aminoacyl-tRNA. After binding, GTP is hydrolyzed and EF-Tu is released. EF-Tu is then regenerated by another elongation factor EF-Ts.

Peptide bond formation

In this step, a peptide bond is formed between the amino acid attached to the tRNA in the P site and the amino acid attached to the tRNA in the A site. This reaction is catalyzed by peptidyl transferase, an enzymatic activity of the 23S rRNA in the large subunit. As a result, the growing polypeptide chain is transferred from the P site to the A site.

Translocation

In this step, the ribosome moves along the mRNA by one codon, shifting the tRNAs from their previous positions. The deacylated tRNA in the P site moves to the E site and exits the ribosome. The peptidyl-tRNA in the A site moves to the P site. The A site becomes vacant and ready for a new aminoacyl-tRNA. This movement requires elongation factor EF-G and GTP, which bind to the ribosome and induce a conformational change.

Elongation continues until a stop codon is reached on the mRNA.

Termination

Termination is the final stage of translation, where protein synthesis stops and the completed polypeptide is released from the ribosome. Termination occurs when one of the three stop codons (UAA, UAG or UGA) enters the A site of the ribosome.

In prokaryotes, termination involves the following steps:

• One of two release factors (RF-1 or RF-2) recognizes the stop codon and binds to the A site. RF-1 recognizes UAA and UAG, while RF-2 recognizes UAA and UGA.
• Another release factor (RF-3) assists RF-1 or RF-2 by binding to GTP and interacting with the ribosome.
• The release factors trigger peptidyl transferase to cleave the bond between the polypeptide and the tRNA in the P site, releasing the polypeptide from the ribosome.
• The mRNA and the tRNAs are also released from the ribosome.
• The ribosome dissociates into its subunits with the help of ribosome recycling factor (RRF) and EF-G.

Before an amino acid can be incorporated into a protein, it must be attached to a specific tRNA molecule that recognizes the codon for that amino acid. This process is called synthesis of aminoacyl-tRNA or tRNA charging.

The enzyme that catalyzes this reaction is called aminoacyl-tRNA synthetase. There are 20 different aminoacyl-tRNA synthetases, one for each amino acid. Each synthetase recognizes only one amino acid and its corresponding tRNAs.

The reaction involves two steps:

1. The amino acid reacts with ATP to form an aminoacyl-adenylate (or aminoacyl-AMP) intermediate and releases pyrophosphate (PPi). This step requires energy from ATP hydrolysis.
2. The aminoacyl group of the aminoacyl-adenylate is transferred to the 3` end of the tRNA, forming an aminoacyl-tRNA and releasing AMP. This step is driven by the high energy bond between the aminoacyl group and AMP.

The overall reaction can be summarized as:

Amino acid + ATP + tRNA → aminoacyl-tRNA + AMP + PPi

The synthesis of aminoacyl-tRNA is also called amino acid activation because it activates the amino acid for peptide bond formation. The activated amino acid has a high energy bond with the tRNA that can be easily broken by the ribosome to join the growing polypeptide chain.

The accuracy of this process is crucial for protein synthesis. If an incorrect amino acid is attached to a tRNA, it will be incorporated into the protein at the wrong position, potentially altering its structure and function. To prevent this, the aminoacyl-tRNA synthetases have two mechanisms of proofreading:

• The first mechanism is called activation-site editing. It occurs during the first step of the reaction, when the synthetase rejects any amino acid that does not fit into its active site.
• The second mechanism is called hydrolytic editing. It occurs after the second step of the reaction, when the synthetase removes any incorrectly attached amino acid from the tRNA by hydrolysis.

These mechanisms ensure that only correctly charged tRNAs enter the translation process.

The first step in protein synthesis is the formation of a complex between the mRNA and the small (30S) ribosomal subunit. This complex is called the 30S initiation complex. The formation of this complex requires a specific sequence in the mRNA called the Shine–Dalgarno sequence, and several proteins called initiation factors (IFs).

The Shine–Dalgarno sequence is a short stretch of nucleotides that is rich in purines (A and G). It is located about 5 to 10 nucleotides upstream of the start codon (AUG) that codes for the first amino acid of the protein. The Shine–Dalgarno sequence is complementary to a part of the 16S rRNA in the small ribosomal subunit. This allows the mRNA and the ribosome to align properly and ensures that the start codon is positioned in the correct site (P site) of the ribosome.

The initiation factors are proteins that facilitate the binding of the mRNA and the ribosome, and also help to recruit the initiator tRNA that carries the first amino acid. In prokaryotes, there are three initiation factors: IF-1, IF-2 and IF-3. The roles of these factors are as follows:

• IF-1 binds to the A site of the small ribosomal subunit and prevents it from binding to other tRNAs or large ribosomal subunits before mRNA binding.
• IF-2 binds to the initiator tRNA (fMet-tRNAfMet) and GTP and delivers them to the P site of the small ribosomal subunit. The initiator tRNA carries a modified form of methionine called N-formylmethionine (fMet), which is unique to prokaryotes. The fMet-tRNAfMet base pairs with the start codon (AUG) on the mRNA.
• IF-3 binds to the E site of the small ribosomal subunit and prevents it from associating with the large ribosomal subunit before mRNA binding.

The formation of the 30S initiation complex involves several steps:

1. The small ribosomal subunit binds to IF-1 and IF-3, which prevent premature association with other components.
2. The small ribosomal subunit binds to the mRNA via base pairing between the Shine–Dalgarno sequence and the 16S rRNA. The small ribosomal subunit scans along the mRNA in a 5` to 3` direction until it reaches the start codon (AUG).
3. The fMet-tRNAfMet/IF-2/GTP complex binds to the start codon in the P site of the small ribosomal subunit. This causes IF-3 to be released from the E site.
4. The large (50S) ribosomal subunit joins the complex, with the release of IF-1 and IF-2 and hydrolysis of GTP. This forms a 70S initiation complex, which is ready for elongation.

The initiation phase of protein synthesis is complete when a 70S initiation complex is formed. The next phase is elongation, where amino acids are added one by one to form a polypeptide chain.

Elongation is the process of adding amino acids to the growing polypeptide chain by reading the mRNA codons one by one. Elongation consists of three steps: aminoacyl-tRNA binding, peptide bond formation and translocation.

Aminoacyl-tRNA binding

The second codon in the mRNA is positioned in the A site of the ribosome. The corresponding aminoacyl-tRNA, which carries the amino acid that matches the codon, binds to the A site via codon–anticodon interaction. This binding requires an elongation factor called EF-Tu and a molecule of GTP. The EF-Tu/GTP complex binds to the aminoacyl-tRNA and delivers it to the A site. After binding, GTP is hydrolyzed and EF-Tu is released.

EF-Tu is then regenerated by another elongation factor called EF-Ts. EF-Ts binds to EF-Tu and exchanges GDP for GTP. The EF-Tu/GTP complex is now ready to bind another aminoacyl-tRNA.

Peptide bond formation

The next step is the formation of a peptide bond between the amino acids in the P site and the A site. This reaction is catalyzed by an enzyme called peptidyl transferase, which is part of the large ribosomal subunit. Peptidyl transferase cleaves the bond between the amino acid and the tRNA in the P site and transfers it to the amino group of the amino acid in the A site. As a result, a dipeptide is formed and attached to the tRNA in the A site.

Translocation

The final step is the movement of the ribosome along the mRNA by one codon. This step requires another elongation factor called EF-G and a molecule of GTP. The EF-G/GTP complex binds to the ribosome and induces a conformational change that causes three simultaneous movements:

• The tRNA in the P site moves to the E site and exits the ribosome.
• The tRNA in the A site moves to the P site with its attached dipeptide.
• The ribosome moves along the mRNA by three nucleotides, bringing a new codon into the A site.

During this process, GTP is hydrolyzed and EF-G is released.

The elongation cycle repeats until a stop codon reaches the A site. Each cycle adds one amino acid to the C-terminal end of the polypeptide chain. The rate of elongation in prokaryotes is about 15-20 amino acids per second.

The final stage of translation is termination, which occurs when a stop codon (UAA, UAG, or UGA) is encountered in the A site of the ribosome. Unlike other codons, there is no tRNA that recognizes these codons. Instead, one of two release factors (RF-1 or RF-2) binds to the A site, depending on the specific stop codon . RF-1 recognizes UAA and UAG, while RF-2 recognizes UAA and UGA . A third release factor, RF-3, assists the interaction of RF-1 or RF-2 with the ribosome .

The binding of the release factor triggers the peptidyl transferase activity of the ribosome to transfer the polypeptide chain from the tRNA in the P site to a water molecule, effectively cleaving the bond between the polypeptide and the tRNA . This releases the newly synthesized polypeptide from the ribosome, followed by the mRNA and the uncharged tRNAs . The ribosome then dissociates into its 30S and 50S subunits, which can be reused for another round of translation .

The termination of translation is summarized in the table below:

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