Topic 7.3 - Translation

polysome

A group of two or more ribosomes translating an mRNA sequence simultaneously

ATP is required to supply energy to link the tRNA with its specific amino acid via a covalent bond. The formation of this covalent bond conserves energy (originally from ATP), which is then used to join amino acids via peptide bonds (at the ribosome). The reaction occurs in two steps: 1. Activation of the amino acid. ATP and the specific amino acid bind to the tRNA activating enzyme. ATP is hydrolysed and the amino acid is covalently linked to AMP. 2. Attachment of amino acid to tRNA. The specific tRNA molecule then binds to the active site. The amino acid is covalently linked to the tRNA and AMP is released. The charged tRNA (tRNA with amino acid) is released from the active site.

Describe how amino acids and tRNA molecules are linked.

Initiation involves the assembly of translation machinery: The small subunit of the ribosome binds to the 5' end of the mRNA. The anticodon (UAC) of an initiator tRNA (carrying methionine) binds to the start codon (AUG) on the mRNA by hydrogen bonds. Anticodon to codon binding follows complementary base pair rules. The initiator tRNA assists in the binding of the large subunit of the ribosome to the small subunit. The initiator tRNA is in the P site of the large subunit.

Describe the events during the initiation of translation.

The primary structure of a protein is the number and sequence of amino acids that make up the linear polypeptide. The number and sequence of amino acids is determined by the base sequence of the gene. The primary structure will determine how the polypeptide will fold-up following translation. The shape of a protein determines its function.

Describe the primary structure of a polypeptide.

Some proteins are comprised of more than one polypeptide. Intermolecular bonds (including hydrogen bonds, ionic bonds, disulfide bridges, and hydrophobic interactions) hold different polypeptide chains together. The number and positioning of polypeptide chains is called quaternary structure. For example, the structural protein collagen is formed by three polypeptide chains winding around each other. Quaternary structure may also include a prosthetic group. A prosthetic group is a non-protein molecule tightly-bound to the protein. For example, the heme group. Hemoglobin is composed of four polypeptides (two alpha and two beta chains). Each polypeptide has a heme group. Proteins containing a prosthetic group are termed conjugated proteins.

Describe the quaternary structure of a protein.

Secondary structure represents the first level of protein folding. It involves hydrogen bonds forming between polar C=O and N−H groups within the polypeptide backbone. These groups are positioned on either side of each peptide bond. There are two types of folding that contribute to secondary structure: In both cases, hydrogen bonds stabilize the structure. Alpha helices and beta-pleated sheets are found in nearly all proteins.

Describe the secondary structure of a polypeptide.

Ribosomes are composed of rRNA and proteins. There are two subunits: a large and a small subunit. On the small subunit, there is a mRNA binding site. On the large subunit, there are three tRNA binding sites: A site - the tRNA carrying the next amino acid to be added to the polypeptide chain binds here. P site - the tRNA with the growing polypeptide chain binds here. E site - the tRNA that has lost its amino acid, exits the ribosome from here

Describe the structure of a ribosome.

tRNA molecules have a stem-loop structure. The stem regions are double stranded due to hydrogen bonding between complementary base pairs. There are three loops, where there is no base pairing. One loop is called the anticodon loop and contains the three base anticodon sequence. There is an amino acid attachment site at the 3' end of the tRNA molecule.

Describe the structure of a tRNA molecule.

Once the translation machinery has been assembled, the polypeptide chain can be synthesized. Polypeptide chain synthesis, or elongation, involves a repeated cycle of events: A charged tRNA binds at the A site. To bind at the A site, the anticodon of the tRNA must be complementary to the codon on the mRNA. A peptide bond forms between the amino acids at the P and A sites (the energy required is provided by the charged tRNA). The tRNA at the P site detaches from its amino acid. The ribosome moves one codon along the mRNA (5' to 3'). The tRNA with the growing polypeptide chain is now in the P site. The tRNA with no amino acid is now in the E site and exits the ribosome (to be recharged by tRNA activating enzyme). A newly charged tRNA can now enter the A site.

Describe the synthesis of a polypeptide chain.

Once the translation machinery has been assembled, the polypeptide chain can be synthesized. Polypeptide chain synthesis, or elongation, involves a repeated cycle of events: A charged tRNA binds at the A site. To bind at the A site, the anticodon of the tRNA must be complementary to the codon on the mRNA. A peptide bond forms between the amino acids at the P and A sites (the energy required is provided by the charged tRNA). The tRNA at the P site detaches from its amino acid. The ribosome moves one codon along the mRNA (5' to 3'). The tRNA with the growing polypeptide chain is now in the P site. The tRNA with no amino acid is now in the E site and exits the ribosome (to be recharged by tRNA activating enzyme). A newly charged tRNA can now enter the A site.

Describe the synthesis of a polypeptide chain.

The ribosome moves down the mRNA until there is a stop codon in the A site. No tRNA molecules can bind to stop codons. Proteins called release factors bind to the A site. This causes: The polypeptide chain to be released from the tRNA in the P site. The ribosome separates from the mRNA and splits into large and small subunits.

Describe the termination of translation.

The tertiary structure is the final, three dimensional shape of a polypeptide. R groups determine the way the polypeptide chain folds up. If surrounded by water, amino acids with hydrophobic (non-polar) R groups tend to be located in the center of the protein and those with hydrophilic (polar or charged) R groups tend to be on the outside. There are four types of interactions between R groups that hold the tertiary structure: Hydrogen bonds form between amino acids with polar R groups. Ionic bonds form between amino acids with oppositely charged R groups. Disulfide bridges (covalent bonds) can form between two amino acids containing sulfur in the R group. Hydrophobic interactions form between amino acids with non-polar R groups.

Describe the tertiary structure of a polypeptide.

Primary (1º) Structure

Determine which protein structure this is.

Quaternary (4º) Structure

Determine which protein structure this is.

Secondary (2º) Structure

Determine which protein structure this is.

Tertiary (3º) Structure

Determine which protein structure this is.

Proteins synthesized on free ribosomes function within the cytoplasm. Free ribosomes are also found in mitochondria and chloroplasts. Proteins synthesized by ribosomes associated with the RER typically have three fates: To be secreted (via exocytosis). For example: Antibodies. To be plasma membrane proteins. For example: Voltage-gated potassium ion channels. To function in membrane-bound organelles. For example: Digestive enzymes within lysosomes.

Distinguish between proteins synthesized on free ribosomes and those synthesized on ribosomes associated with the RER.

1. mRNA binds to the small subunit of the ribosome 2. The small subunit of the ribosome moves along the mRNA molecule in a 5' to 3' direction until it reaches a start codon (AUG) 3. The small subunit of the ribosome moves along the mRNA molecule in a 5' to 3' direction until it reaches a start codon (AUG) 4. The large subunit of the ribosome binds to the tRNA and small subunit 5. A second tRNA (with amino acid attached) complementary to the second codon on the mRNA then binds to the P site of the ribosome 6. The amino acid carried by the tRNA in the P site is transferred to the amino acid in the A site as a consequence of the ribosome catalyzing a new peptide bond (condensation reaction). The growing polypeptide increases in length. 7. The ribosome moves one codon along the mRNA (in a 5' - 3' direction): The tRNA in the P site is moved to the E site and then released The tRNA in the A site is moved into P site 8. Another tRNA binds, complementary to the next codon on the mRNA, binds to the A site. 9. Steps 6, 7, and 8 are repeated until a stop codon is reached. 10. When a stop codon is reached translation is stopped: a release factor attaches to the A site the polypeptide chain is released the ribosome complex dissembles ready for reuse translating another mRNA molecule

Explain the 10 step process of translation

In eukaryotes, the ribosomes are separated from the genetic material (DNA and RNA) by the nucleus. After transcription, the mRNA must be transported from the nucleus (via nuclear pores) prior to translation by the ribosome. This transport requires modification to the RNA construct (e.g. 5'-methyl capping and 3'-polyadenylation). Prokaryotes lack compartmentalised structures (like the nucleus) and so transcription and translation need not be separated. Ribosomes may begin translating the mRNA molecule while it is still being transcribed from the DNA template. This is possible because both transcription and translation occur in a 5' → 3' direction

Explain the difference between the timing of translation in eukaryotes and prokaryotes.

A second tRNA molecule pairs with the next codon in the ribosomal A site. The amino acid in the P site is covalently attached via a peptide bond (condensation reaction) to the amino acid in the A site. The tRNA in the P site is now deacylated (no amino acid), while the tRNA in the A site carries the peptide chain

Explain the steps of elongation in translation

The first stage of translation involves the assembly of the three components that carry out the process (mRNA, tRNA, ribosome). The small ribosomal subunit binds to the 5'-end of the mRNA and moves along it until it reaches the start codon (AUG). Next, the appropriate tRNA molecule bind to the codon via its anticodon (according to complementary base pairing). Finally, the large ribosomal subunit aligns itself to the tRNA molecule at the P site and forms a complex with the small subunit

Explain the steps of initiation in translation

The final stage of translation involves the disassembly of the components and the release of a polypeptide chain. Elongation and translocation continue in a repeating cycle until the ribosome reaches a stop codon. These codons do not recruit a tRNA molecule, but instead recruit a release factor that signals for translation to stop. The polypeptide is released and the ribosome disassembles back into its two independent subunits

Explain the steps of termination in translation

The ribosome moves along the mRNA strand by one codon position (in a 5' → 3' direction). The deacylated tRNA moves into the E site and is released, while the tRNA carrying the peptide chain moves to the P site. Another tRNA molecules attaches to the next codon in the now unoccupied A site and the process is repeated

Explain the steps of translocation in translation

tRNA molecules transfer amino acids to the ribosome. tRNA molecules differ in their anticodon sequence. Different anticodons correspond to different amino acids. The anticodon determines the order in which amino acids are added to the growing polypeptide chain (at the ribosome). The correct amino acid must be added to the tRNA. This is carried out by tRNA activating enzymes. There are 20 different, specific tRNA activating enzymes, one for each of the 20 different amino acids. The active site of the tRNA activating enzyme, binds its specific amino acid, the specific tRNA, and ATP. Differences in amino acid structure and anticodon sequences allow for specificity.

How does a tRNA molecule ensure the genetic code is correctly translated into amino acid sequence?

The ribosome remains free and unattached

What happens to proteins when they are targeted for intracellular use within the cytosol?

The ribosome becomes bound to the ER

What happens to proteins when they are targeted for secretion, membrane fixation or use in lysosomes

The SRP-ribosome complex docks at a receptor located on the ER membrane (forming rough ER). Translation is re-initiated and the polypeptide chain continues to grow via a transport channel into the lumen of the ER. The synthesised protein will then be transported via a vesicle to the Golgi complex (for secretion) or the lysosome. Proteins targeted for membrane fixation (e.g. integral proteins) get embedded into the ER membrane. The signal sequence is cleaved and the SRP recycled once the polypeptide is completely synthesised within the ER.

What is the Role of the Signal Recognition Particle in Protein Destination?

The presence or absence of an initial signal sequence on a nascent polypeptide chain. The presence of this signal sequence results in the recruitment of a signal recognition particle (SRP), which halts translation

What is the protein destination determined by?

These processes work together to create a polypeptide which in turns folds to become a protein. Proteins carry many essential functions in cells. For more detail review 2.4.U7 Living organisms synthesize many different proteins with a wide range of functions.

What is the purpose of transcription and translation?

The structures seen are called polysomes. Polysomes are formed by several ribosomes translating a single mRNA. Electron micrograph 1 shows polysomes in a eukaryotic cell. Electron micrograph 2 shows several polysomes associated with a single gene, revealing simultaneous transcription and translation in prokaryotes (they do not have a nucleus). In prokaryotes, ribosomes can attach to the 5' end of the mRNA molecule as soon as transcription starts.

What structures can be identified in the following electron micrographs?