Protein Synthesis: tRNA Charging and Ribosome Structure

Aminoacyl tRNA Synthetase

• An enzyme that charges tRNA
• Each tRNA synthetase only binds to one specific tRNA and amino acid
• The amino acid fits perfectly in a space on the enzyme, and the tRNA also fits in a specific spot

Charging tRNA

• tRNA must be "charged" with an attached amino acid to participate in protein synthesis
• tRNA charging requires energy

Ribosome Structure

• A large subunit with three binding sites:
  • A (aminoacyl tRNA) site: binds the anticodon of charged tRNA
  • P (peptidyl tRNA) site: where the tRNA with the growing polypeptide chain resides
  • E (exit) site: where the uncharged tRNA exits

Translation

• Three steps:
  • Initiation: the small ribosomal subunit binds to mRNA and the initiator charged tRNA, forming an initiation complex
  • Elongation: new amino acids are added to the growing polypeptide chain
  • Termination: the process stops when a stop codon is reached

Initiation

• The small ribosomal subunit binds to mRNA and the initiator charged tRNA
• In prokaryotes, the small subunit binds to the Shine-Dalgarno sequence on mRNA
• In eukaryotes, the small subunit binds to the 5' cap
• The start codon is AUG
• The first amino acid is always methionine, which can be removed after translation

Elongation

• A new charged tRNA enters the A site, requiring energy
• The large subunit catalyzes the transfer of the growing polypeptide chain from the P site to the A site, which does not require energy
• The uncharged tRNA moves to the E site, and the tRNA with the growing chain moves to the P site, requiring energy

Termination

• The ribosome encounters a stop codon
• A release factor binds to the stop codon and triggers the release of the polypeptide chain
• The ribosome dissociates from the mRNA

DNA Replication

• DNA is replicated semi-conservatively, each new strand contains one old strand and one new strand
• Replication occurs at a replication fork, where the DNA strands are separated
• The leading strand is synthesized continuously, in the same direction as the unwinding of the DNA
• The lagging strand is synthesized discontinuously, in the opposite direction from the unwinding of the DNA, forming Okazaki fragments

Okazaki Fragments

• Short DNA fragments that are synthesized on the lagging strand
• Named after Reiji Okazaki

Replacing Primer Fragments

• DNA polymerase III adds nucleotides to the 3' end of the new primer, until reaching the primer of the previous fragment
• DNA polymerase I then replaces the primer with DNA
• DNA ligase then joins the newly synthesized DNA fragments

Replication Termination

• In prokaryotes, Topoisomerase IV decatenates the newly replicated DNA from the old DNA

Mutations

• Heritable changes in DNA sequences
• Can have beneficial, harmful, or neutral effects on the protein

Spontaneous Mutations

• Occur without outside influence
• Can be caused by internal chemical reactions that alter bases
• Example: deamination of cytosine to uracil

Induced Mutations

• Caused by external mutagens
• Example: radiation

Transcription Initiation

• Occurs at specific DNA sequences called promoters
• RNA polymerase binds to the promoter and starts transcription, creating an RNA copy of the DNA

Prokaryotic Gene Expression

• Prokaryotic genes are often organized into operons
• Operon: a gene cluster with a single promoter, including:
  • A promoter
  • Two or more structural genes
  • An operator

Transcription Factors

• Regulatory proteins that bind to the operator and affect transcription
• Activators increase transcription
• Repressors decrease transcription

Lac Operon

• Inducible system that is only turned on when lactose is present
• When lactose is absent, the repressor binds to the operator and prevents transcription
• When lactose is present, lactose binds to the repressor, changing its shape and preventing it from binding to the operator
• This allows RNA polymerase to bind to the promoter and transcribe the lac operon genes

RNA Processing

• In eukaryotes, pre-mRNA is processed before it is translated
• Introns (noncoding regions) are removed
• Exons (coding regions) are spliced together

Central Dogma of Molecular Biology

• Describes the flow of genetic information in a cell
• DNA is transcribed to RNA
• RNA is translated to protein

Codons and Translation

• Three nucleotides (a codon) code for a specific amino acid
• The start codon defines the reading frame
• Shifts in the reading frame will change the codons and amino acids specified

Types of Mutations

• Silent mutation: a base substitution that results in a codon for the same amino acid
• Missense mutation: a base substitution that results in a codon for a different amino acid
• Nonsense mutation: a base substitution that results in a stop codon
• Frameshift mutation: an insertion or deletion of a base pair that alters the reading frame
• Loss-of-stop mutation: a base substitution that changes a stop codon to a sense codon

Different Types of RNA

• mRNA (messenger RNA): carries genetic information from DNA to ribosomes
• tRNA (transfer RNA): carries amino acids to the ribosome
• rRNA (ribosomal RNA): forms part of the ribosome structure

tRNA Structure

• tRNA has a cloverleaf structure
• One end has an anticodon that can base pair with a specific mRNA codon
• The other end binds to an amino acid