mRNA Translation and Amino Acids

• Translation occurs after transcription and involves using a mature mRNA molecule as a template for protein synthesis.

Molecular Components of Translation

• The 20 amino acids that are available in cell cytoplasm are essential for translation.
• Each of the various transfer RNA (tRNA) types connect a messenger RNA (mRNA) codon to its matching amino acid.
• Ribosomes add each amino acid that is carried by a tRNA to the elongating end of a polypeptide chain.

Amino Acid Monomers

• Amino acids are organic molecules that possess both carboxyl and amino groups.
• Properties differ because of differing side chains, known as R groups.
• Physical and chemical properties of the R group define an amino acid's properties.
• The 20 different amino acids are the building blocks of proteins.
• Amino acids can contain hydrophobic R groups.
• Amino acids can contain hydrophilic R groups.
• Amino acids can contain functional groups that are charged at a cell's specific pH.

tRNA

• Transfer RNA (tRNA) molecules are not all identical.
• Each tRNA molecule consists of about 80 nucleotides folding into a three-dimensional structure.
• tRNA includes an anticodon site, and an attachment site to carry a specific amino acid.
• The anticodon triplet is unique to each tRNA type.
• The anticodon base-pairs with a messenger RNA (mRNA)'s complementary codon.
• There are 45 types of tRNA to carry 20 amino acids.
• Some tRNAs anticodons recognize more than one codon.
• Rules for base pairing between the third base of the codon and anticodon are relaxed, and called "wobble".
• An example of "wobble" shows that at the wobble position, guanine (G) on the anticodon can bind with cytosine (C) or uracil (U) in the third position of a codon.
• "wobble" explains why synonymous codons for an amino acid can differ in their third base, but not usually in other bases.

Aminoacyl-tRNA Synthetase

• A cell translates an mRNA message into protein with the help of tRNA.
• Each amino acid is joined to the correct tRNA by aminoacyl-tRNA synthetase.
• There are 20 different aminoacyl-tRNA synthetases.
• Each aminoacyl-tRNA synthetase has active sites for only a specific tRNA and amino acid combination.
• Aminoacyl-tRNA synthetase joins each amino acid to the correct tRNA.
• A synthetase enzyme's active site attach to the amino acid and ATP.
• ATP loses two phosphate groups and joins the amino acid as AMP.
• Appropriate tRNA covalently bonds to amino acid, displacing AMP.
• Activated amino acid is released as Aminoacyl tRNA ("activated amino acid").

The Ribosome

• Each ribosome consists of a large and small subunit.
• Subunits are composed of proteins and ribosomal RNA (rRNA).
• Ribosomal (r) proteins are made by translation in the cytoplasm.
• Ribosomal (r) RNA is generated by transcription in the nucelus.
• The arrangement of r-proteins and rRNA into large ribonucleoprotein complexes needs dedicated mechanisms to ensure their co-localization.
• R-proteins need to be delivered to the nucleus where they assemble with the rRNA.
• Eukaryotic ribosomes have a large 60S and a small 40S subunit, each with ribosomal RNA (rRNA) and ribosomal proteins
• Mammalian ribosomes have an 80s size.
• Prokaryotic and eukaryotic ribosomes differ.
• A ribosome has an mRNA binding site and three tRNA binding sites (A, P, and E sites).
• The P site (peptidyl-tRNA binding site) holds the tRNA carrying the growing polypeptide chain.
• The A site (aminoacyl-tRNA binding site) carries the tRNA with the next amino acid to be added to the chain.
• The E site (exit site) is where discharged tRNAs leave the ribosome.
• rRNA rather than protein carries out a ribosome's functions, this is supported by recent advances.
• Ribosomal (r)RNA is the main constituent at the interphase between the two subunits and the A and P sites.
• A ribosome is the catalyst for peptide bond formation.

Building a Polypeptide

• Translation is divided into three stages: initiation, elongation, and termination.
• Both initiation and chain elongation require energy provided by the hydrolysis of GTP.

Initiation of Translation

• A small ribosomal subunit binds with messenger RNA (mRNA) and a special initiator tRNA, which carries methionine and attaches to the start codon.
• Proteins called initiation factors bring in the large subunit.
• The arrival of a large ribosomal subunit completes the initiation complex.

The Elongation Cycle of Translation

• Elongation has a series of three-step cycles as each amino acid is added to the preceding one.
  • Codon recognition - the anticodon of an incoming aminoacyl tRNA base-pairs with the complementary messenger RNA (mRNA) codon in the A site, the hydrolysis of GTP increases the accuracy and efficiency of this step.
  • Peptide bond formation - an rRNA molecule of the large subunit catalyzes formation of a peptide bond between the new amino acid in the A site and the carboxyl end of the growing polypeptide in the P site, this step attaches the polypeptide to the tRNA in the A site.
  • Translocation - the ribosome translocates the tRNA in the A site to the P site, the empty tRNA in the P site is moved to the E site where it is released, the messenger RNA (mRNA) moves along with its bound tRNAs, and the next codon is translated into the A site.
• Amino acids are linked by peptide bonds.
• Repeating this process creates a polypeptide chain.

Termination of Translation

• Termination happens when one of the three stop codons reaches the A site.
• A release factor binds to the stop codon and hydrolyzes the bond between the polypeptide and its tRNA in the P site.
• This frees the polypeptide, and the translation complex disassembles.

Polyribosomes

• A single messenger RNA (mRNA) is used to make many copies of a polypeptide simultaneously.
• A number of ribosomes can translate a single messenger RNA (mRNA) molecule simultaneously, forming a polyribosome.
• Polyribosomes exist in prokaryotic and eukaryotic cells.

Summary of Transcription and Translation in Eukaryotic Cells

• RNA is transcribed from a DNA template.
• In eukaryotes, the RNA transcript (pre-mRNA) is spliced and modified to produce mRNA that moves from the nucleus to the cytoplasm.
• After leaving the nucleus, mRNA attaches to the ribosome.
• Each amino acid attaches to its appropriate tRNA with the help of a specific enzyme and ATP.
• A succession of tRNAs add their amino acids to a polypeptide chain as the mRNA is moved through the ribosome one codon at a time.

Free vs Bound Ribosomes

• Two populations of ribosomes are evident in cells as ''free'' and ''bound''.
• Free ribosomes stay suspended in the cytosol, and synthesize proteins that reside in the cytosol.
• Bound ribosomes stay attached to the cytosolic side of the endoplasmic reticulum.
• Although bound and free ribosomes are identical in structure, their location depends on the type of protein that they produce.
• Translation begins in the cytosol for all ribosomes.
• A polypeptide targeted for the endomembrane system or for export has a specific signal peptide.
• This signal peptide contains a sequence of about 20 amino acids.
• A signal recognition particle (SRP) binds to the signal peptide, and then attaches both to a receptor protein in the ER membrane, haltering synthesis momentarily.
• The signal recognition particle (SRP) consists of a protein-RNA complex.
• The SRP leaves after binding, and protein synthesis resumes with the growing polypeptide snaking across the membrane into the cisternal space via a protein pore.
• An enzyme usually cleaves the signal polypeptide.
• Secretory proteins are released entirely into the cisternal space, but membrane proteins are partially embedded in the ER membrane.
• Other signal peptides are used to target polypeptides to mitochondria, chloroplasts, the nucleus, or other organelles that are not part of the endomembrane system.
• In these cases, translation is completes in the cytosol before the polypeptide is imported into the organelle.

Comparing Protein Synthesis in Prokaryotes and Eukaryotes Differences

• Prokaryotes and eukaryotes have have differences in cellular machinery and in details of the processes of transcription/translation, but are similar.
• Eukaryotic RNA polymerases differ from those of prokaryotes and require transcription factors.
• They differ in how transcription is terminated.
• Their ribosomes also are different.
• Gene expression comparison reveals key differences.
• Prokaryotic cells lack a nuclear envelope.
  • This allows translation to begin while transcription is still in progress.
• In eukaryotes, the nuclear envelope segregates transcription from translation.
• Extensive RNA processing is carried out between these processes in eukaryotes.
• Eukaryotic cells also have complicated mechanisms for targeting proteins to the appropriate organelle.