Protein Synthesis and Translation Notes

Protein Production and Synthesis

Goal

The goal is to produce a protein from the instructions on a gene.

DNA and Complementarity

• DNA consists of two strands due to the complementarity of nucleotides.
• Typically, only one strand of a gene is the sense-making strand (coding strand).
• The other strand serves as the template strand for transcription.

Transcription

• Transcription involves making an RNA complement of the DNA gene.
• Enzyme: RNA polymerase.
• RNA polymerase reads the DNA molecule in the 3' to 5' direction and builds the new RNA strand in the 5' to 3' direction.
• Transcription starts and stops at specific points on the DNA.
• After transcription, the DNA closes back up, remaining protected, while the temporary RNA copy (recipe card) is used for translation.

Translation

• Translation is the process of reading the RNA molecule and creating a protein based on its instructions.
• Codons: Each set of three nucleotides on the RNA molecule.
• A protein is composed of amino acids in a specific sequence (primary sequence).

Codons and Amino Acids

• Scientists determined the relationship between codons and amino acids in the 1960s and 70s.
• There are three positions in a nucleotide, with four different nucleotides possible at each position.
• 4^3 = 64 possible combinations of codons.
• There are about 20 different amino acids commonly found in proteins.
• Some codons are redundant, meaning multiple codons can code for the same amino acid.

The Genetic Code Chart

• The genetic code chart shows which amino acid each codon specifies.
• Example: AUG codes for methionine (MET).
• Each three-letter abbreviation (e.g., VAL, ALA, LYS) represents an amino acid.

Process of Translation

• The ribosome reads the mRNA and matches each codon to its corresponding amino acid.
• Example: CAG codes for glutamine (GLN).

Example Problem

Given a DNA sequence (coding strand) and transcribing and translating it to polypeptide sequence.

Key Concepts for the problem for transcription

• In RNA, uracil (U) replaces thymine (T).
• AUG is typically the start codon, but it may occurs elsewhere
• Certain codons (stop codons) signal the end of the polypeptide chain (e.g., UAA, UAG, UGA).

Wobble Room

• The third nucleotide in a codon is often less important for determining the amino acid.
• Geneticists call this "wobble room" or "genetic wobble."
• The last codon is degenerate meaning that it doesn't matter to some degree.
• A mutation in the third position may not always affect the resulting protein sequence.

Reversing the Process

• It's possible to work backward from an amino acid sequence to an RNA sequence and then to a DNA sequence.
• If multiple codons can code for the same amino acid, any of those codons can be used.
• Codons start with the 5' end.

tRNA (Transfer RNA)

• tRNA molecules are transcribed from tRNA genes.
• They fold up on themselves due to complementary regions.
• Stem loops: structure on tRNA.

Anticodon

• Anticodon: A three-nucleotide sequence on the tRNA that is complementary to the mRNA codon.
• Example: UAC is complementary to AUG.
• Each tRNA molecule carries a specific amino acid corresponding to its anticodon.
• There are 61 different types of tRNA, each with a unique anticodon.
• tRNA molecules deliver amino acids to the site of translation.

Ribosomes

• Ribosomes are the machines that perform translation.
• Ribosomes have two subunits: a large subunit and a small subunit.
• There are three sites in the ribosome: E (exit), P (polypeptide), and A (aminoacyl tRNA binding site).

Process in the Ribosome

1. tRNA molecules, loaded with their corresponding amino acids, enter the A site of the ribosome.
2. If the anticodon on the tRNA matches the codon on the mRNA, the tRNA stays in the A site.
3. A covalent (peptide) bond forms between the amino acid on the tRNA in the A site and the growing polypeptide chain, which is attached to the tRNA in the P site.
4. The ribosome moves (translocates) three nucleotides down the mRNA (Jink sound).
5. The tRNA that was in the P site moves to the E site and is ejected from the ribosome.
6. The tRNA that was in the A site moves to the P site.
7. A new tRNA enters the A site, and the process repeats.
8. When the ribosome reaches a stop codon, a release factor protein binds to the stop codon.
9. The ribosome disassembles, releasing the polypeptide.

Enzymes for tRNA

• Aminoacyl tRNA synthetase enzymes: Enzymes that reload each tRNA with the correct amino acid.
• These enzymes require ATP to covalently bond the amino acid to the tRNA.

Prokaryotes Versus Eukaryotes

• In bacteria (prokaryotes), there is no nucleus, so transcription and translation happen in the cytoplasm.
• In eukaryotic cells, transcription happens in the nucleus, and translation happens in the cytoplasm (on the rough ER).
• In prokaryotes, ribosomes can start translating the mRNA even before transcription is complete.

Eukaryotic mRNA Processing

• In eukaryotic cells, the initial RNA transcript is called pre-mRNA.
• Pre-mRNA undergoes processing before it can be translated:
  • A 5' guanine cap is added to the 5' end of the mRNA.
  • A 3' poly-A tail (a string of adenine nucleotides) is added to the 3' end.
  • Introns (non-coding regions) are removed from the mRNA.
  • Exons (coding regions) are spliced together to form the final mRNA molecule.

Spliceosomes and Snurps

• Spliceosomes: Molecular machines in the nucleus that remove introns from pre-mRNA.
• Spliceosomes are made of small nuclear ribonucleoproteins (snRNPs or snurps) and other proteins.
• Spliceosomes loop the intron into a lariat shape and cut it out, then ligate the exons together.

Alternative Splicing

• Alternative splicing: A process in which different combinations of exons are spliced together, resulting in multiple different mRNA molecules from a single gene.
• Allows one gene to encode multiple variants of a protein.
• Does not occur in prokaryotes.
• Increases the diversity of proteins that can be produced from a limited number of genes.

Example

• Gene, which serves as recipe, for chocolate chip cookies.
• Alternate splicing can take out instructions such as "add a half a cup of crushed walnuts" allowing the gene to have different outcomes.

Comparisons:

• E. coli can make 1000 polypeptides from 1000 genes.
• Human genome can produce greater than 100,000 genes from 22,000 genes because of alternative splicing.