🧬Biochemistry Unit 9 – Protein Synthesis

Key Concepts and Terminology

• Central dogma of molecular biology describes the flow of genetic information from DNA to RNA to proteins
• Nucleotides are the building blocks of DNA and RNA consisting of a sugar, phosphate group, and nitrogenous base (adenine, guanine, cytosine, thymine in DNA; uracil in RNA)
• Codons are triplets of nucleotides that code for specific amino acids during translation
  • 64 possible codons with 61 coding for amino acids and 3 serving as stop codons (UAA, UAG, UGA)
• Ribosomes are the sites of protein synthesis composed of rRNA and proteins
  • Contain a large and small subunit that assemble during translation
• Genetic code is the set of rules that defines the relationship between codons and amino acids
  • Universal across all organisms with a few exceptions (mitochondrial genetic code)
• Promoters are DNA sequences upstream of genes that initiate transcription by binding RNA polymerase
• Shine-Dalgarno sequence is a ribosomal binding site located upstream of the start codon in prokaryotic mRNA

DNA Structure and Function

• DNA is a double-stranded helix composed of nucleotides held together by hydrogen bonds between complementary base pairs (A-T, G-C)
  • Strands are antiparallel with a 5' to 3' directionality
• DNA serves as the genetic blueprint for an organism containing the instructions for protein synthesis
• Genes are segments of DNA that code for specific proteins or functional RNA molecules
• DNA replication is the process of creating an identical copy of the DNA molecule during cell division
  • Ensures genetic information is passed on to daughter cells
• DNA packaging involves wrapping DNA around histone proteins to form nucleosomes and higher-order chromatin structures
  • Allows for compact storage of DNA within the nucleus
• DNA damage can occur due to various factors (UV radiation, chemicals) and is repaired by DNA repair mechanisms to maintain genomic integrity

Transcription Process

• Transcription is the synthesis of RNA from a DNA template catalyzed by RNA polymerase
• Initiation involves the binding of RNA polymerase to the promoter region and unwinding of the DNA double helix
  • Transcription factors assist in the assembly of the transcription initiation complex
• Elongation proceeds as RNA polymerase moves along the DNA template strand in the 5' to 3' direction adding nucleotides to the growing RNA chain
  • RNA polymerase catalyzes the formation of phosphodiester bonds between nucleotides
• Termination occurs when RNA polymerase encounters a termination signal causing the release of the newly synthesized RNA and dissociation of the polymerase
• In eukaryotes, transcription occurs in the nucleus and the resulting pre-mRNA undergoes processing before translation
• Prokaryotic transcription and translation are coupled with translation often beginning before transcription is complete

RNA Processing and Modification

• Pre-mRNA undergoes several modifications before becoming mature mRNA ready for translation
• 5' capping adds a 7-methylguanosine cap to the 5' end of the pre-mRNA protecting it from degradation and facilitating translation initiation
• 3' polyadenylation adds a poly(A) tail to the 3' end of the pre-mRNA increasing stability and aiding in export from the nucleus
• Splicing removes introns (non-coding regions) and joins exons (coding regions) to create a continuous coding sequence
  • Spliceosome, a complex of snRNPs and proteins, catalyzes the splicing reaction
• Alternative splicing allows for the production of multiple protein isoforms from a single gene by selective inclusion or exclusion of exons
• RNA editing modifies specific nucleotides in the RNA sequence (adenosine to inosine, cytidine to uridine) altering the amino acid sequence of the resulting protein
• Mature mRNA is exported from the nucleus to the cytoplasm for translation

Translation Mechanism

• Translation is the synthesis of proteins from an mRNA template occurring on ribosomes
• Initiation involves the assembly of the translation initiation complex at the start codon (AUG) of the mRNA
  • Eukaryotic initiation factors (eIFs) and met-tRNAi facilitate ribosome assembly and positioning
• Elongation proceeds as the ribosome moves along the mRNA in the 5' to 3' direction adding amino acids to the growing polypeptide chain
  • tRNAs deliver amino acids to the ribosome based on codon-anticodon recognition
  • Peptidyl transferase catalyzes the formation of peptide bonds between amino acids
• Termination occurs when the ribosome encounters a stop codon (UAA, UAG, UGA) leading to the release of the newly synthesized polypeptide and dissociation of the ribosome
  • Release factors (RF1, RF2, RF3) recognize stop codons and facilitate termination
• Polyribosomes (polysomes) are formed when multiple ribosomes simultaneously translate the same mRNA molecule allowing for efficient protein synthesis

Protein Structure and Folding

• Proteins are linear polymers of amino acids that fold into specific three-dimensional structures
• Primary structure is the linear sequence of amino acids in a protein determined by the genetic code
• Secondary structure refers to the local folding of the polypeptide chain into alpha helices and beta sheets stabilized by hydrogen bonds
  • Ramachandran plot depicts the allowed phi and psi angles for secondary structure formation
• Tertiary structure is the overall three-dimensional shape of a protein resulting from interactions between secondary structure elements
  • Stabilized by hydrophobic interactions, hydrogen bonds, ionic bonds, and disulfide bridges
• Quaternary structure involves the assembly of multiple polypeptide subunits into a functional protein complex (hemoglobin, DNA polymerase)
• Protein folding is guided by the amino acid sequence and aided by molecular chaperones that prevent misfolding and aggregation
  • Misfolded proteins can lead to diseases (Alzheimer's, Parkinson's)

Regulation of Protein Synthesis

• Protein synthesis is tightly regulated to ensure proper cellular function and respond to environmental changes
• Transcriptional regulation controls the rate of mRNA synthesis by modulating the activity of RNA polymerase
  • Transcription factors bind to regulatory sequences (enhancers, silencers) to activate or repress transcription
  • Chromatin modifications (histone acetylation, DNA methylation) affect the accessibility of DNA to transcription machinery
• Post-transcriptional regulation involves the processing, stability, and localization of mRNA molecules
  • microRNAs (miRNAs) and RNA-binding proteins (RBPs) can destabilize mRNA or inhibit translation
• Translational regulation controls the rate of protein synthesis by modulating the efficiency of translation initiation or elongation
  • Phosphorylation of translation initiation factors (eIF2α) can inhibit global protein synthesis during stress conditions
• Post-translational modifications (phosphorylation, glycosylation, ubiquitination) can alter protein function, stability, and localization
• Feedback inhibition allows end products of a metabolic pathway to inhibit the activity of enzymes involved in their own synthesis

Applications and Relevance in Biochemistry

• Understanding protein synthesis is crucial for developing treatments for genetic diseases caused by mutations in genes or regulatory elements
  • Gene therapy aims to introduce functional copies of genes to compensate for defective ones
• Recombinant DNA technology allows for the production of proteins in heterologous hosts (bacteria, yeast) for research and therapeutic purposes
  • Insulin, growth hormones, and antibodies are produced using recombinant DNA methods
• Protein engineering involves modifying the amino acid sequence of proteins to alter their properties or create novel functions
  • Directed evolution and rational design are used to create proteins with enhanced stability, specificity, or catalytic activity
• Studying the regulation of protein synthesis provides insights into the molecular basis of diseases (cancer, metabolic disorders) and helps identify potential drug targets
  • Inhibitors of transcription factors or translation initiation are being explored as anticancer agents
• Proteomics aims to characterize the entire complement of proteins expressed in a cell or organism under specific conditions
  • Mass spectrometry and protein microarrays are used to analyze protein abundance, interactions, and modifications
• Protein structure determination techniques (X-ray crystallography, NMR spectroscopy, cryo-EM) provide atomic-level details of protein folding and function
  • Enables structure-based drug design and understanding of disease-causing mutations