Translation in Bacteria

Introduction

• Translation involves decoding mRNA and covalently linking amino acids together to form a polypeptide; this occurs at the ribosome.
• Translation begins when a ribosome binds mRNA and is positioned properly so that translation will yield the correct amino acid sequence in the polypeptide chain.
• Transfer RNA molecules carry amino acids to the ribosome so that they can be added to the polypeptide chain as the ribosome moves down the mRNA molecule.
• Just as DNA and RNA synthesis proceeds in one direction, so too does protein synthesis. Polypeptide synthesis begins with the amino acid at the end of the chain with a free amino group (the N-terminal) and moves in the C-terminal direction. Thus, translation occurs in the amino terminus to the carboxyl terminus direction.
• Protein synthesis is accurate and very rapid. In E.coli, synthesis occurs at a rate of at least 900 amino acids added per minute.
• Cells that grow quickly must use each mRNA with great efficiency to synthesize proteins at a sufficiently rapid rate.
• To achieve rapid rates of protein synthesis, mRNAs often are simultaneously complexed with several ribosomes, each ribosome reading the mRNA message and synthesizing a polypeptide.
• At maximal rates of mRNA use, there may be a ribosome every 80 nucleotides along the mRNA or as many as 20 ribosomes simultaneously reading an mRNA that codes for a 50,000-dalton polypeptide.
• A complex of mRNA with several ribosomes is called a polyribosome or polysome. Polysomes are present in all organisms.
• Bacteria can further increase the efficiency of gene expression by coupling transcription and translation.
• While RNA polymerase is synthesizing an mRNA, ribosomes can already be attached to the mRNA so that transcription and translation occur simultaneously.
• Coupled transcription and translation are possible in bacterial cells because a nuclear envelope does not separate the translation machinery from DNA, as it does in eukaryotes.

Amino Acid Activation

• Amino acid activation is a preparatory step for protein synthesis.
• A ready supply of tRNA molecules bearing the correct amino acid is necessary for translation to occur.
• Transfer RNA (tRNA) molecules are about 70 to 95 nucleotides long and possess several characteristic structural features.
• When folded, tRNA assumes a cloverleaf conformation with an acceptor stem holding the activated amino acid.
• The 3′ end of all tRNAs has the same CCA sequence, and in all cases, the amino acid is attached to the A nucleotide.
• The anticodon is complementary to an mRNA codon and is located on the anticodon arm.
• Aminoacyl-tRNA synthetases catalyze amino acid activation and are specific for a single amino acid and its tRNAs.
• The amino acid is attached to the tRNA by a high-energy bond, and the energy stored in this bond provides the fuel needed to generate the peptide bond when the amino acid is added to the growing peptide chain.
• Proofreading by some aminoacyl-tRNA synthetases ensures that the correct amino acid is attached to the corresponding tRNA, as an incorrect amino acid will be incorporated into a polypeptide in place of the correct amino acid.
• The protein synthetic machinery recognizes only the anticodon of the amino acyl-tRNA and cannot tell whether the correct amino acid is attached.

Ribosomes Have three tRNA Binding Sites

• Protein synthesis occurs on ribosomes, with mRNA serving as the blueprint.
• Ribosomes are formed from two subunits, the large and small subunits, and contain rRNA molecules and polypeptide chains.
• Ribosomes can be divided into two domains: translational and exit domains.
• The translational domain interacts with tRNAs and is responsible for forming peptide bonds.
• Three tRNA binding sites are found within the translational domain: A, P, and E sites.
• The A site receives tRNAs carrying an amino acid to be added to the protein.
• The P site holds a tRNA attached to the growing polypeptide.
• The E site is the location from which empty tRNAs leave the ribosome.
• Ribosomal RNA (rRNA) has three roles in protein synthesis.
• The 16S rRNA of the 30S subunit is needed for the initiation of protein synthesis.
• The 16S rRNA binds to the Shine-Dalgarno sequence on mRNA and helps position the mRNA on the ribosome.
• The 16S rRNA also binds initiation factor 3 and the 39 CCA end of amino-acyl-tRNA.
• The 23S rRNA is a ribozyme that catalyzes peptide bond formation.

Protein Synthesis Begins with Formation of the 70S Initiation Complex

• Protein synthesis is divided into three stages: initiation, elongation, and termination.
• Initiation of protein synthesis in bacteria begins with a modified aminoacyl tRNA called N-formyl methionyl-treatment (fMet-tRNA), which is the initiator tRNA coded for by the start codon AUG.
• The amino acid of the initiator tRNA has a formyl group covalently bound to the amino group and can be used only for initiation because of the presence of the formyl group.
• Protein synthesis in bacteria starts with the formation of the 30S initiation complex, consisting of the initiator tRNA, the mRNA to be translated, and the 30S ribosomal subunit; two initiation factors (IF-1 and IF-2) are involved.
• Positioning of the initiator fMet-tRNA on the mRNA is crucial for proper translation of the mRNA.
• The 16S rRNA within the 30S subunit helps in aligning the Shine-Dalgarno sequence in the leader sequence of the mRNA with the AUG codon to ensure that the start codon will be translated first.
• Once the 30S initiation complex is formed, it binds the 50S ribosomal subunit, forming the 70S initiation complex.
• The fMet-tRNA is positioned at the peptidyl or P site, where it begins the synthesis of the polypeptide chain.
• The third initiation factor (IF-3) prevents the 30S and 50S subunits from binding each other earlier in the initiation stage.
• GTP is used as a high-energy molecule to accomplish initiation.
• Translation initiation of mRNAs lacking a Shine-Dalgarno sequence in the leader or lacking leaders altogether is still unclear.

Elongation of the Polypeptide Chain

• Elongation of the polypeptide chain is a complex process that involves three phases: amino acyl-tRNA binding, the transpeptidation reaction, and translocation.

Amino acyl-tRNA binding phase:

• This is the first phase of the elongation cycle.
• Proteins called elongation factors (EF) aid the process.
• An amino acid corresponding to the proper mRNA codon is added to the C-terminal end of the polypeptide chain.
• The ribosome moves down the mRNA in the 59 to 39 direction.
• The P site is filled with either the initiator fMet-tRNA or a tRNA bearing a growing polypeptide chain.
• The A and E sites are empty.
• Messenger RNA is bound to the ribosome in such a way that the proper codon interacts with the P site tRNA (e.g., an AUG codon for fMet-tRNA).
• The next codon is located within the A site and is ready to accept an aminoacyl-tRNA.
• In bacterial cells, this phase is aided by two elongation factors and requires the expenditure of one GTP.

Transpeptidation reaction phase:

• This is the second phase of the elongation cycle.
• Transpeptidation is catalyzed by the peptidyl transferase activity of the 23S rRNA ribozyme, which is part of the 50S ribosomal subunit.
• In this reaction, the amino group of the A-site amino acid reacts with the carboxyl group of the C-terminal amino acid on the P-site tRNA.
• This results in the transfer of the peptide chain from the tRNA in the P site to the tRNA in the A site, as a peptide bond is formed between the peptide chain and the incoming amino acid.
• No extra energy source is required for peptide bond formation because the bond linking an amino acid to tRNA is high in energy.

Translocation phase:

• This is the final phase in the elongation cycle.
• Three things happen simultaneously: the peptidyl-tRNA moves from the A site to the P site, the ribosome moves one codon along mRNA, and the empty tRNA moves from the P site to the E site and subsequently leaves the ribosome.
• Translocation involves rotations of the 30S and 50S subunits relative to each other.
• The head portion of the 30S subunit swivels.
• These changes in ribosome structure move the tRNAs into their new locations; the codon-anticodon interactions between the tRNAs and the mRNA moves the mRNA as the tRNAs move.
• One elongation factor participates and one GTP is hydrolyzed during this intricate process.

Insertion of Selenocysteine and Pyrrolysine

• Two unusual amino acids, selenocysteine, and pyrrolysine, can be inserted during translation.
• Selenocysteine is synthesized from serine and recognized by a specific elongation factor before being incorporated into the growing polypeptide chain.
• Selenocysteine is inserted at a UGA stop codon that is located near a sequence element called SECIS.
• SECIS is found immediately after the UGA stops the codon in bacteria.
• Pyrrolysine, on the other hand, is synthesized from lysine before being attached to a specific tRNA with a CUA anticodon.
• The pyrrolysine-tRNA is recognized by a specific aminoacyl-tRNA synthetase and inserted at UAG stop codons located near a sequence element called PYLIS.
• Both SECIS and PYLIS form stem-loop structures that prevent cessation of translation.

Protein Synthesis Ends When the Ribosome Reaches a Stop Codon

• Protein synthesis stops when the ribosome reaches a stop codon, which can be UAA, UAG, or UGA.
• The stop codon is located on the mRNA immediately before the trailer.
• Three release factors (RF-1, RF-2, and RF-3) help the ribosome recognize the stop codon.
• Since no corresponding tRNA exists for a stop codon, the ribosome ceases protein synthesis.
• Peptidyl transferase hydrolyzes the bond between the polypeptide and the tRNA in the P site, releasing the polypeptide and the empty tRNA.
• During this process, GTP hydrolysis takes place.
• The ribosome then dissociates from the mRNA and separates into the 30S and 50S subunits.
• IF-3 binds the 30S subunit, which prepares it for the next round of protein synthesis.

Protein synthesis is an energy-expensive process that requires multiple high-energy bonds and GTP hydrolysis.

Ensuring Accuracy During Translation

• The high energy expenditure is required to ensure the fidelity of protein synthesis.
• Fidelity is assessed before and after the formation of the peptide bond.
• Correct pairing of the anticodon and codon causes conformational changes in the ribosome components, which facilitate peptide bond formation.
• If an incorrect aminoacyl-tRNA enters the A site, the tRNA is ejected.
• Rarely, an incorrect aminoacyl-tRNA is selected, and a peptide bond is formed between the wrong amino acid and the growing polypeptide.
• The ribosome can detect this error, but it’s unclear how.
• Release factors recognize the presence of an incorrect amino acid, leading to hydrolysis of the aberrant polypeptide from the tRNA, its release from the ribosome, and termination of translation.