COPY: Translation, tRNA and Ribosomes

Translation

• Translation is the process where mRNA directs the synthesis of proteins from amino acids.
• This requires mRNA, tRNA with attached amino acid, and ribosomes.

Transfer RNAs (tRNAs)

• tRNAs are short, single-stranded RNA molecules, that are about 74 to 95 nucleotides long.
• Each tRNA carries one particular amino acid.
• Base pairing between the tRNA anticodon and mRNA codon determines where an amino acid is incorporated into a growing polypeptide.

tRNA Structure

• Anticodons are read from 5' to 3'.
• tRNAs can carry modified bases that are produced by the chemical alteration of A, G, C, and U nucleotides.

Genetic Code

• There are 61 codons for amino acids, excluding stop codons.
• 61 unique tRNA genes are not required.
• Inosine (I) recognizes A, U, or C.

Wobble

• Some tRNAs recognize more than one codon due to a phenomenon known as wobble
• Remember to use the wobble rules

Ribosomes

• Ribosomes are the site of protein synthesis.
• They have a complex structure composed of protein and RNA, with large and small subunits.

Ribosome Subunits

• Subunit sizes differ between prokaryotes and eukaryotes.
• Prokaryotic small subunits carry 16S rRNA
• Eukaryotic ribosomes have 18S rRNA.

Translation Initiation in E. coli

• The Shine-Dalgarno sequence in E. coli is recognized by complimentary sequences in the 16S rRNA of the 30S subunit.
• A downstream AUG codon signals the initiation of translation by adding a tRNA carrying formylmethionine.
• Some bacteria use GUG (valine) as a start codon instead of AUG (e.g., Mycobacterium tuberculosis).
• The large 50S ribosomal subunit binds such that the tRNAFMet is placed in the P site of the ribosome, which completes the initiation of translation.

Elongation Phase in E. coli

• Elongation factors (EFs) escort the next tRNA into the A site of the ribosome.
• Peptidyl transferase catalyzes the formation of a peptide bond between the carboxyl (C) terminus of formylmethionine and the amino (N) terminus of the second amino acid.
• As the ribosome moves, fMet moves from the P site to the E site.
• A new tRNA enters the A site, fMet is released, and the process repeats.

Termination Phase in E. coli

• A nonsense (stop) codon is encountered, for which there is no tRNA.
• The release factor recognizes the stop codon and moves into the A site.
• The polypeptide is released from the C-terminal tRNA.
• The mRNA, tRNA, and ribosomal subunits dissociate from each other.

Translation Initiation in Eukaryotes

• The 40S ribosome subunit recognizes and binds the 5' methylated cap.
• It then scans along the mRNA until it reaches the initiation sequence.
• In many eukaryotes, this is only AUG, but includes surrounding sequences in mammals.
• The initiator tRNA carries Met, not fMet.
• The rest of translation is similar to prokaryotes.

Polyribosomes

• A polyribosome is a complex of several ribosomes translating from the same mRNA.

Prokaryotes vs Eukaryotes

• Prokaryotes lack cellular organelles.
• DNA replication, transcription, and translation all occur in the cytoplasm and are tightly coupled in prokaryotes.
• Eukaryotes have membrane-bound organelles and compartmentalization.
• In eukaryotes, DNA replication and transcription occur in the nucleus, and translation occurs in the cytoplasm (ER), hence it is uncoupled.
• In prokaryotes, translation can begin as soon as enough of the transcript is made, and mRNA is very short-lived (seconds).
• In eukaryotes, mRNA must travel out of the nucleus to the cytoplasm before being translated, and mRNA is longer-lived (minutes to hours).

Post-Translational Processing

• These are modifications that occur to the protein after translation

Transcription

• Transcription creates a RNA copy from a DNA sequence

RNA Polymerase

• RNA polymerase is the enzyme that catalyzes transcription.
• Promoters are DNA sequences near the beginnings of genes that signal RNA polymerase where to begin transcription.
• Terminators are sequences in the RNA products that tell RNA polymerase where to stop and are encoded by DNA.
• Promoters are not start codons, and terminators are not stop codons.

Transcription Steps in E. coli

• During initiation, the sigma (σ) factor recognizes the promoter.
• RNA polymerase doesn't require a primer.
• During elongation, the DNA helix reforms, displacing the RNA transcript.
• Termination can be Rho-dependent or Rho-independent.

Rho-Dependent Transcription Termination

• Rho protein binds to an RNA sequence that is C-rich/G-poor without secondary structure

Rho-Independent Transcription Termination

• No protein involved, a GC-rich RNA region makes hairpin structure

DNA Replication vs Transcription

• Transcription uses DNA replication to create a RNA sequence

Prokaryotic Genes

• What you see is what you get; mRNA and proteins can be directly deduced from the DNA sequence
• Template strand / antisense strand: 3’ to 5’
• RNA-like strand / sense strand / coding strand: 5’ to 3’

Eukaryotic Genes

• A gene that codes for a protein has:
  • Gene (DNA)
  • Transcription
  • Primary RNA transcript (still in nucleus)
  • RNA processing
  • Spliced RNA (in cytoplasm)
  • 5' cap
  • poly-A tail
  • and is finally translated

Eukaryotic mRNA Ends

• Addition of methylated cap at the 5’ end: The capping enzyme adds a guanidine triphosphate in reverse orientation to the 5' end after polymerization of the transcript’s first few nucleotides (NOT encoded by gene)
• Addition of 100-200 adenosine's to the 3' end, known as the poly-A tail (NOT encoded by the gene)

Eukaryotic Genes: Introns

• Not all eukaryotic genes contain introns, some contain multiple introns
• RNA splicing removes introns from a primary transcript

RNA Splicing

• RNA splicing usually is carried out by a complex known as the spliceosome, although some RNA transcripts are self-splicing

Introns

• Allow for alternative splicing:
• RNA produces different mature mRNA molecules from one gene -May encode related proteins with different, partially overlapping sequences

Alternative Splicing

• Alternative splicing may contribute to the complexity of mammals

Genome Size

• Polychaos dubium has 670,000,000,000 bp, marbled lungfish has 130,000,000,000 bp.
• Humans have 3,000,000,000 bp E. coli has 4,600,000 bp.