Concept 17.4 Translation is the RNA-directed synthesis of a polypeptide: a closer look

· In the process of translation, a cell translates a genetic message to build a polypeptide.

The tRNA molecule is a translator.

· The interpreter is transfer RNA (tRNA), which transfers amino acids from the cytoplasmic pool to a growing polypeptide in a ribosome.

○ A cell has all 20 amino acids available in its cytoplasm, either by synthesizing them from scratch or by taking them up from the

surrounding solution.

· Each type of tRNA molecule translates a particular mRNA codon into a particular amino acid. Each tRNA molecule bears a specific

amino acid at one end. At the other end of the tRNA is a nucleotide triplet called an anfile:///Users/reem.albitar/Desktop/Screen%20Shot%202015-05-12%20at%2012.20.05%20PM.pngticodon, which base-pairs with a complementary

codon on mRNA. { fig. 17.15 The structure of transfer RNA (tRNA)}.

· Codon by codon, the genetic message is translated as tRNAs deposit amino acids in the order prescribed, and the ribosome joins the amino acids into a chain. tRNA is a translator because it reads a nucleic acid word (the mRNA codon) and interprets it as a protein word (the amino acid).

· Like other types of RNA, tRNA molecules are transcribed from DNA templates.

· In both bacterial and eukaryotic cells, each tRNA is used repeatedly, picking up its designated amino acid in the cytosol, depositing the amino acid at the ribosome, and returning to the cytosol to pick up another copy of that amino acid.

· A tRNA molecule consists of a single strand of about 80 nucleotides folded back on itself to form a three-dimensional structure. tRNA includes a loop containing the anticodon and an attachment site at the 3¢ end for an amino acid.

· Each amino acid is joined to the correct tRNA by aminoacyl-tRNA synthetase.

○ The 20 different synthetases match the 20 different amino acids.

○ Each has active sites for only a specific tRNA–amino acid combination.

· The synthetase catalyzes a covalent bond between them in a process driven by ATP hydrolysis.

○ The result is an aminoacyl-tRNA or charged amino acid.

· The second recognition process involves a correct match between the tRNA anticodon and an mRNA codon.

· If each anticodon had to be a perfect match to each codon, we would expect to find 61 types of tRNA, but the actual number is about 45, because the anticodons of some tRNAs recognize more than one codon.

· Such versatility is possible because the rules for base pairing between the third base of the codon and the anticodon are relaxed. This flexible base pairing is called wobble.

○ Wobble explains why the synonymous codons for a given amino acid most often differ in their third base, not in their other bases.

The ribosome is the site of translation.

· Ribosomes facilitate the specific coupling of the tRNA anticodons with mRNA codons during protein synthesis.

A ribosome consists of a large and a small subunit, each made up of proteins and ribosomal RNA (rRNA).

· In eukaryotes, the subunits are made in the nucleolus. rRNA genes are transcribed and the RNA is processed and assembled with proteins imported from the cytoplasm. The subunits are exported via nuclear pores to the cytoplasm.

· In both bacteria and eukaryotic cells, large and small subunits join to form a functional ribosome only when they attach to an mRNA molecule.

○ Because most cells contain thousands of ribosomes, rRNA is the most abundant type of cellular RNA.

· Though similar in structure and function, bacterial and eukaryotic ribosomes have enough differences that certain antibiotic drugs (like tetracycline and streptomycin) can inactivate bacterial ribosomes without inhibiting eukaryotic ribosomes.

· Each ribosome has a binding site for mRNA and three binding sites for tRNA molecules.

○ The P site (peptidyl tRNA-binding site) holds the tRNA carrying the growing polypeptide chain.

○ The A site (aminoacyl tRNA-binding site) holds the tRNA carrying the next amino acid to be added to the chain.

○ Discharged tRNAs leave the ribosome at the E (exit) site.

· The ribosome holds the tRNA and mRNA in close proximity and positions the new amino acid for addition to the carboxyl end of the growing polypeptide.

○ It then catalyzes the formation of the peptide bond.

· As the polypeptide becomes longer, it passes through an exit tunnel in the ribosome’s large unit and is released to the cytosol through the exit tunnel.

· Evidence supports the hypothesis that rRNA, not protein, is responsible for the ribosome’s structure and function.

○ Proteins on the exterior support the shape changes of the rRNA molecules as they carry out catalysis during translation.

○ rRNA is the main constituent at the interface between the two subunits and of the A and P sites, and it is the catalyst for peptide bond formation.

○ A ribosome can be regarded as one colossal ribozyme.

The process of translation builds a polypeptide.

· Translation can be divided into three stages: initiation, elongation, and termination.

○ All three phases require protein “factors” that aid in the translation process.

○ Both initiation and chain elongation require energy provided by the hydrolysis of GTP.

· Initiation brings together mRNA, a tRNA with the first amino acid, and the two ribosomal subunits.

· First, a small ribosomal subunit binds with mRNA and a special initiator tRNA, which carries methionine.

○ In bacteria, the binding occurs at a specific RNA sequence, just upstream of the start codon, AUG.

○ In eukaryotes, the small subunit, with the initiator tRNA already bound, binds to the 5¢ cap of the mRNA and then moves, or scans, downstream along the mRNA until it reaches the start codon AUG, which signals the start of translation.

○ This establishes the codon reading frame for the mRNA.

· The union of mRNA, initiator tRNA, and a small ribosomal subunit is followed by the attachment of a large ribosomal subunit, forming the translation initiation complex.

○ Proteins called initiation factors bring all these components together.

○ Energy in the form of a GTP molecule is invested in the formation of the initiation complex.

· In the elongation stage of translation, amino acids are added one by one to the previous amino acid at the C-terminus of the growing chain.

○ Each addition involves the participation of protein elongation factors and occurs in three-step cycles as each amino acid is added to the preceding one.

· Energy expenditure occurs in the first and third steps.

○ Codon recognition requires hydrolysis of one molecule of GTP, which increases the accuracy and efficiency of this step.

○ One more GTP is hydrolyzed to provide energy for the translocation step.

· The mRNA is moved through the ribosome in one direction only, 5¢ end first; this is equivalent to the ribosome moving 5¢ ® 3¢ on the mRNA.

○ The ribosome and the mRNA move relative to each other, unidirectionally, codon by codon.

· The elongation cycle takes less than a tenth of a second in bacteria and is repeated as each amino acid is added to the chain until the polypeptide is completed.

o The empty tRNAs that are released from the E site return to he cytoplasm, where they will be reloaded with the appropriate amino acid.

· Termination occurs when one of the three stop codons reaches the A site of the ribosome.

○ A release factor binds to the stop codon and causes hydrolysis of the bond between the polypeptide and its tRNA in the P site.

○ This frees the polypeptide, which is released through the exit tunnel of the ribosome’s large subunit.

· The translation complex disassembles.

○ Breakdown of the translation assembly requires the hydrolysis of two more GTP molecules.

· A ribosome requires less than a minute to translate an average-sized mRNA into a polypeptide.

· A single mRNA may be used to make many copies of a polypeptide simultaneously as multiple ribosomes, polyribosomes or polysomes, trail along the same mRNA.

○ Polyribosomes can be found in bacterial and eukaryotic cells.

Folding and modification of a protein follows translation.

· During and after synthesis, a polypeptide spontaneously coils and folds to its three-dimensional shape.

○ The primary structure, the order of amino acids, determines the secondary and tertiary structure.

· A chaperone protein (chaperonin) helps the polypeptide fold correctly.

· In addition, proteins may require post-translational modifications before doing their particular job.

· These modifications may require additions such as sugars, lipids, or phosphate groups to amino acids.

○ Enzymes may remove one or more amino acids from the leading (amino) end of the polypeptide chain.

· In some cases, a single polypeptide chain may be enzymatically cleaved into two or more pieces.

○ In other cases, two or more polypeptides may join to form a protein with quaternary structure.

Signal peptides target some eukaryotic polypeptides to specific destinations in the cell.

· Two populations of ribosomes, free and bound, are active participants in protein synthesis.

○ Free ribosomes are suspended in the cytosol and synthesize proteins that reside and function in the cytosol.

○ Bound ribosomes are attached to the cytosolic side of the endoplasmic reticulum (ER) or to the nuclear envelope.

○ Bound ribosomes make proteins of the endomembrane system as well as proteins secreted from the cell.

· Although bound and free ribosomes are identical in structure, their location depends on the type of protein they are synthesizing.

· Translation in all ribosomes begins in the cytosol, but a polypeptide destined for the endomembrane system or for export has a specific signal peptide region at or near the leading end.

○ The signal peptide consists of a sequence of about 20 amino acids.

· A signal recognition particle (SRP) binds to the signal peptide and attaches it and its ribosome to a receptor protein in the ER membrane.

○ The SRP, which consists of a protein–RNA complex, functions as an escort to bring the ribosome to a receptor protein built into the ER membrane.

○ The receptor is part of a multiprotein translocation complex.

· Protein synthesis resumes, with the growing polypeptide snaking across the membrane into the ER lumen via a protein pore.

○ An enzyme usually cleaves the signal polypeptide.

· Secretory proteins are released into solution within the ER lumen, but membrane proteins remain partially embedded in the ER membrane.

· Other kinds of signal peptides are used to target polypeptides to mitochondria, chloroplasts, the nucleus, and other organelles that are not part of the endomembrane system.

○ In these cases, translation is completed in the cytosol before the polypeptide is imported into the organelle.

○ While the mechanisms of translocation vary, each of these polypeptides has a “zip code” that ensures its delivery to the correct cellular location.

· Bacteria also employ signal sequences to target proteins to the plasma membrane for secretion.

· Although bacteria and eukaryotes carry out transcription and translation in similar ways, they differ in cellular machinery and in the details of the processes.

· Bacterial and eukaryotic RNA polymerases differ significantly from each other.

· Transcription is terminated differently in bacteria and eukaryotes.

· Bacterial and eukaryotic ribosomes differ slightly.

· Initiation of translation is slightly different in bacteria and eukaryotes.

· Gene expression in eukaryotes differs from that of bacteria because of the greater compartmental organization of the eukaryotic cell.

○ In the absence of a nucleus, a bacterial cell can simultaneously transcribe and translate the same gene and the new protein quickly diffuses to its operating site.

· In eukaryotes, the nuclear envelope segregates transcription from translation and provides a compartment for extensive RNA processing between these processes.

○ This provides additional steps whose regulation helps coordinate the elaborate activities of a eukaryotic cell.

	Created by Reema Al Biatr over 11 years ago

Describe the stages of protein synthesis