Module 2.3: Translation

Initiation factors

Help assemble trasnlational machinery

Elongation Factors

• deliver tRNAs

• help to move ribosomes along the mRNA

Release factors

Helps terminat translation and release the completed polypeptide

Aminoacyl-tRNA synthetases

Attach correct AA to their corresponding tRNAs

Ribosomal RNA and Proteins

• For the ribosomes where translation occurs

• ensure that genetic message is accurately converted into a polypeptide

Transfer RNA (tRNA)

• Recognizes mRNA codons and carries the corresponding AA to ribosomes, where they are added to the growing polypeptide chain

• converts genetic information in mRNA into a polypeptide

Structure of tRNA

• consist of single RNA strand: approx 70-90 codons long

• Complementary regions in the tRNA strand from hydrogen bonds, producing 4 double-helical structures and 3 loops.

in 2- dimensions: clover leaf, in 30 dimensions: L-shaped

Anticodon

• sequence of 3 nucleotides that pairs with complementary codon on the mRNA

• mRNA codons = 5” - 3” direction, while tRNA pairs with them in the antiparallel (3’ - 5’)

AA Attachment Site

• At the 3” end of the tRNA, there is conserved CCA nucleotide sequence.

• The adenine at the end is the attachment site for AA

Aminocylation / tRNA charging

• Process of attaching an AA to tRNA, resulting in the tRNA being called CHARGED

• resulting molecules is called aminoacyl-tRNA

Aminoacyl-tRNA Synthetases

• Each synthetase recognizes a particular amino acid and its corresponding tRNA or group of tRNAs.

• 20 aminoacyl-tRNA synthetases, corresponding to the 20 standard amino acids.

• catalyze the attachment of an amino acid to its tRNA using energy from ATP.

Aminoacyl-tRNA

• delivers its specific amino acid to the ribosome for incorporation into a growing polypeptide chain.

• the ribosome primarily checks the pairing between the mRNA codon and the tRNA anticodon

• therefore, the aminoacyl-tRNA synthetase is responsible for ensuring that the tRNA carries the correct amino acid.

Genetic Code

• contains 64 possible codond from 20 AA

• degenrate: multiple codons can specify same AA

Wobble Hypothesis

• Some tRNA molecules can recognize more than one codon because pairing at the third nucleotide of the codon is more flexible than pairing at the first two positions.

• this flexibilty of the tRNA is called “Wobble”

• The first nucleotide at the 5’ end of the codon pairs with third nucleotide at the 3’ end of the anticodon

• explains why fewer tRNA species are required to recognize all the codons

Translation

• ribosomes and other cellular components read the genetic message contained in mRNA and convert it into a specific amino acid sequence for a protein

• in Proks: occurs simultaneously with transcription in the cytoplasm

• in euks: transcription and translation are separated by nuclear envelope and occurs in the cytoplasm on both free ribosomes or bound ones to the ER

Translation Initiation Complex

• in both proks and euks, small and large ribosomal subunits assemble into a functional ribosome as part of the translation initiation process.

• other components of initiation include the mRNA to be translated, initiator mRNA carrying methionine in euks, and initiation factors to assemble the complex.

Reading mRNA:

• codons read sequentially as mRNA passes through ribosomes

• Each codon specifies an amino acid or a translation stop signal.

• Charged tRNAs deliver amino acids to the ribosome, where they are joined together to form a growing polypeptide chain.

• Translation continues until the ribosome reaches a stop codon, at which point the polypeptide is released.

Translation Initiation in Euk

1. Recognition of the mRNA: Initiation factors bind to the 5′ cap and help recruit the small ribosomal subunit.

2. Recruitment of the initiator tRNA: The initiator tRNA carrying methionine associates with the small subunit and initiation factors.

3. Scanning: The partially assembled complex moves along the mRNA in the 5′ to 3′ direction and reads it in non-overlapping groups of 3 nucleotides called codons until it encounters an appropriate AUG start codon.

4. Start codon recognition: Recognition of AUG establishes the reading frame.

5. Joining of the large subunit: The large ribosomal subunit joins the complex, with GTP hydrolysis helping drive the required steps and rearrangements.
   
   

Translation Initiation in Prok

• mRNA doesn’t contain 5’ cap

• small ribosomal subunit recognizes binding site called Shine Dalgarno Sequence located upstream of the start codon

Polycistronic mRNA

• Prokaryotes mRNAs can be polycistronic, meaning a single mRNA can contain coding sequences for multiple proteins.

• occurs due to functionally related genes often being grouped together and being transcribed as a single unit with a common promoter

• Each coding region can have its own ribosome-binding site, allowing the ribosome to initiate translation separately for each protein

Elongation:

• Initiation factors are released or dissociate at the beginning of elongation

• Proteins are synthesized from the amino (N) terminus to the carboxyl (C) terminus, meaning the first amino acid in a new polypeptide is usually methionine, although it may later be removed.

• The ribosome reads mRNA in successive, non-overlapping groups of three nucleotides called codons.

• Each incoming charged tRNA is delivered to the ribosome by an elongation factor associated with GTP.

• When the codon and anticodon pair correctly, GTP hydrolysis helps the tRNA become fully accommodated in the ribosome, allowing its amino acid to participate in peptide bond formation.

tRNA binding sites in Ribosomes:

1. A site (aminoacyl site): Accepts the incoming charged tRNA that follow the initiator tRNA.

2. P site (peptidyl site): Holds the tRNA carrying the growing polypeptide; the initiator tRNA is positioned directly in the P site.

3. E site (exit site): Holds the deacylated tRNA briefly before it leaves the ribosome.

Peptide Bond Formation:

• After a charged aminoacyl-tRNA enters the A site, the ribosome catalyzes the formation of a peptide bond.

• ribosomal RNA in the large subunit provides the catalytic activity for this reaction, which is known as peptidyl transferase.

• During peptide bond formation, the growing polypeptide is transferred from the tRNA in the P site to the amino acid attached to the tRNA in the A site.

• The ribosome then moves along the mRNA by one codon. This movement is called translocation and requires elongation factors and energy from GTP hydrolysis.

Translocation Process:

• The tRNA carrying the growing polypeptide moves from the A site to the P site.

• The deacylated tRNA moves from the P site toward the E site.

• The deacylated tRNA exits the ribosome.

• The A site becomes available for the next charged tRNA.

And then this cycle repeats to keep on elongating the polypeptide chain one AA at a time

Translation Termination:

Translation terminates when the ribosome reaches a stop codon on the mRNA.

The three stop codons are:

• UAA, UAG, UGA

Stop codons are not recognized by ordinary aminoacyl-tRNAs.

Release factors bind to the ribosome's A site to promote hydrolysis of the bond connecting the completed polypeptide to the tRNA in the P site.

This releases the newly synthesized polypeptide.

 GTP hydrolysis and other factors help disassemble the translation complex, releasing the ribosomal subunits, mRNA, and remaining bound tRNAs.

Eukaryotic Translation Summarized:

1. Initiation: Initiation factors help recruit small ribosomal subunit and initiator methionine-tRNA to the mRNA. The complex scans for an appropriate AUG start codon, and the large subunit joins.

2. Elongation: Charged tRNAs enter the A site, and the ribosome forms peptide bonds between successive amino acids.

3. Translocation: The ribosome moves along the mRNA, shifting tRNAs between the A, P, and E sites.

4. Termination: When the ribosome reaches a stop codon, release factors promote the release of the completed polypeptide and the disassembly of the translation complex.
   
   

The One-Gene–One-Enzyme Hypothesis:

• In the 1940s, George Beadle and Edward Tatum developed this hypothesis

• The hypothesis was based on the observation that filamentous fungus Neurospora crassa (bread mold) can grow on minimal medium, which contains basic nutrients such as simple sugars, inorganic salts, and essential vitamins.

• To grow under these conditions, the fungus must be able to synthesize many of the molecules it needs, including amino acids and vitamins.

• These biosynthetic processes require enzymes encoded by genes.

The One-Gene–One-Enzyme Hypothesis: Arginine synthesis

• Normal Neurospora cells can grow on medium that does not contain the amino acid arginine because they can synthesize arginine themselves.

• Arginine is produced through a metabolic pathway involving several steps. Each step requires a particular enzyme to convert one compound into the next

• Using an enzyme for each conversion. a precursor → into ornithine → then citrulline → finally arginine.

Testing the One-Gene–One-Enzyme Hypothesis;

Adrian Srb and Norman Horowitz conducted further research to investigate whether specific genes were responsible for producing the enzymes required for arginine synthesis.

1. They exposed Neurospora cells to radiation, which can cause mutations in DNA.

2. They then grew the treated cells on different types of growth media:

• Minimal medium without additional supplements

• Medium supplemented with ornithin

• Medium supplemented with citrulline

• Medium supplemented with arginine

2. By comparing growth under these conditions, they could determine which steps in the arginine biosynthesis pathway were disrupted by mutations.

Results of testing the One-Gene–One-Enzyme Hypothesis;

• Arginine-Supplemented Medium: the fungus continued to grow, serving as a positive control, showing that he cells could still grow when the final product of the pathway was supplied directly, suggesting that the mutations affect the synthesis of arginine rather than the cell’s ability to grow.

• Unsupplemented Medium: some mutants failed to grow, which suggested that radiation had produced mutations affecting genes required for arginine biosynthesis.

• Supplemented with Ornithine or Citrulline: they observed different growth patterns. These results allowed them to identify mutants with defects at different stages of arginine synthesis. This means that if a mutant could grow when a particular AA was supplied and the one before it wasn’t, this means that the defect is likely in the step that produces that given AA.

One-gene–one-polypeptide hypothesis:

This is the newer name for the one-gene-one-Enzyme Hypothesis given to it after researches realized that genes can encode for many types of proteins and not just enzymes

Exceptions to the One-Gene–One-Polypeptide Hypothesis:

• When the human genome was sequenced, researchers identified approximately 20,000 protein-coding genes, far fewer than the total number of distinct proteins and protein forms found in the human body.

• This showed that one gene can contribute to the production of multiple protein products.

Mechanisms That Contribute to the production of multiple proteins via one gene:

1. Alternative Splicing: Different combinations of exons can be joined together to produce different mRNA transcripts from the same gene.

2. Post-translational modifications: Proteins can be chemically modified after translation, changing their activity, stability, location, or interactions.

3. Protein processing: Polypeptides can be cleaved or assembled with other components to produce functional protein forms.