🦠Cell Biology Unit 15 – Translation and Protein Synthesis

Key Concepts and Terminology

• Central dogma of molecular biology describes the flow of genetic information from DNA to RNA to proteins
• Transcription process of synthesizing RNA from a DNA template catalyzed by RNA polymerase enzymes
• Translation process of synthesizing proteins from an mRNA template catalyzed by ribosomes
• Genetic code set of rules that defines the correspondence between codons in mRNA and amino acids in proteins
• Codons triplets of nucleotides in mRNA that specify specific amino acids or stop signals during translation
• Reading frame determines the grouping of codons and affects the amino acid sequence of the translated protein
• Open reading frame (ORF) sequence of codons in mRNA that begins with a start codon and ends with a stop codon

DNA to RNA: Transcription Process

• Transcription initiation begins when RNA polymerase binds to a promoter region on the DNA template strand
• RNA polymerase unwinds the double-stranded DNA and separates the template strand from the coding strand
• Elongation phase RNA polymerase moves along the template strand and synthesizes a complementary RNA strand in the 5' to 3' direction
  • RNA nucleotides (A, U, G, C) are added to the growing RNA strand based on complementary base pairing with the DNA template
  • RNA sugar-phosphate backbone is formed through phosphodiester bonds between nucleotides
• Termination occurs when RNA polymerase reaches a termination signal on the DNA template (hairpin loop or protein factors)
  • RNA polymerase dissociates from the DNA template and releases the newly synthesized RNA transcript
• Primary transcript initial RNA product of transcription that undergoes further processing before becoming a mature RNA

mRNA Processing and Modification

• 5' capping addition of a modified guanine nucleotide (7-methylguanosine) to the 5' end of the pre-mRNA
  • Cap protects mRNA from degradation and facilitates translation initiation by binding to ribosomes
• 3' polyadenylation addition of a poly(A) tail (sequence of adenine nucleotides) to the 3' end of the pre-mRNA
  • Poly(A) tail enhances mRNA stability and facilitates export from the nucleus to the cytoplasm
• Splicing removal of introns (non-coding sequences) and joining of exons (coding sequences) in the pre-mRNA
  • Spliceosome complex of small nuclear RNAs (snRNAs) and proteins catalyzes the splicing reaction
  • Alternative splicing generates multiple mRNA variants from a single gene by selectively including or excluding exons
• Mature mRNA processed mRNA that is ready for translation and contains a 5' cap, coding sequence, and 3' poly(A) tail
• mRNA export mature mRNA is transported from the nucleus to the cytoplasm through nuclear pore complexes

Ribosomes: Structure and Function

• Ribosomes macromolecular complexes responsible for protein synthesis during translation
• Composed of ribosomal RNA (rRNA) and ribosomal proteins that assemble into two subunits (large and small)
• Prokaryotic ribosomes (70S) consist of a 50S large subunit and a 30S small subunit
• Eukaryotic ribosomes (80S) consist of a 60S large subunit and a 40S small subunit
• Ribosomal subunits associate during translation initiation and dissociate after translation termination
• Peptidyl transferase center (PTC) catalytic site in the large ribosomal subunit that forms peptide bonds between amino acids
• Ribosomal binding sites (A, P, E) tRNA binding sites on the ribosome that accommodate aminoacyl-tRNA, peptidyl-tRNA, and deacylated tRNA, respectively

tRNA and Amino Acids

• Transfer RNA (tRNA) adapter molecules that link codons in mRNA to specific amino acids during translation
• Cloverleaf secondary structure of tRNA formed by intramolecular base pairing and contains four main loops (D, anticodon, variable, TΨC)
• Anticodon loop contains a triplet of nucleotides (anticodon) that base-pairs with a complementary codon in mRNA
• Amino acid attachment site (3' end) where a specific amino acid is covalently linked to the tRNA by aminoacyl-tRNA synthetases
• Aminoacyl-tRNA synthetases enzymes that catalyze the attachment of amino acids to their cognate tRNAs in a two-step reaction (activation and transfer)
  • Amino acid activation ATP-dependent formation of an aminoacyl-AMP intermediate
  • Amino acid transfer covalent attachment of the activated amino acid to the 3' end of the tRNA
• Wobble base pairing non-standard base pairing between the third base of a codon and the first base of an anticodon allows for degeneracy in the genetic code

Translation Mechanism and Steps

• Translation initiation assembly of the translation initiation complex on the mRNA
  • Small ribosomal subunit binds to the 5' cap of mRNA and scans for the start codon (AUG)
  • Initiation factors (eIFs in eukaryotes, IFs in prokaryotes) facilitate ribosomal subunit association and positioning at the start codon
  • Initiator tRNA (Met-tRNAi) carrying the amino acid methionine binds to the P site of the ribosome
• Elongation cycle repetitive process of adding amino acids to the growing polypeptide chain
  • Aminoacyl-tRNA enters the A site of the ribosome and base-pairs with the corresponding codon in mRNA
  • Peptide bond formation occurs between the amino acid on the A-site tRNA and the growing polypeptide chain on the P-site tRNA
  • Translocation ribosome moves one codon along the mRNA, shifting the tRNAs from A and P sites to P and E sites, respectively
• Termination recognition of a stop codon (UAA, UAG, UGA) in the A site by release factors (RFs)
  • Release factors promote the hydrolysis of the peptidyl-tRNA bond, releasing the completed polypeptide chain
  • Ribosomal subunits and other components dissociate from the mRNA and are recycled for subsequent rounds of translation

Protein Folding and Modifications

• Protein folding process by which a linear polypeptide chain acquires its native three-dimensional structure
• Primary structure linear sequence of amino acids in a polypeptide chain determined by the genetic code
• Secondary structure regular local structures (α-helices and β-sheets) formed by hydrogen bonding between amino acid residues
• Tertiary structure overall three-dimensional shape of a polypeptide chain resulting from interactions between secondary structures
• Quaternary structure assembly of multiple polypeptide subunits into a functional protein complex
• Chaperones proteins that assist in the folding of other proteins by preventing aggregation and promoting proper folding pathways
• Post-translational modifications (PTMs) covalent modifications of amino acid residues that occur after protein synthesis
  • Examples phosphorylation, glycosylation, acetylation, methylation, and disulfide bond formation
  • PTMs can regulate protein function, stability, localization, and interactions with other molecules

Regulation of Protein Synthesis

• Transcriptional regulation control of gene expression at the level of transcription by transcription factors and regulatory elements (promoters, enhancers, silencers)
• Translational regulation control of protein synthesis at the level of translation by RNA-binding proteins, microRNAs, and regulatory factors
  • Examples ribosome binding site accessibility, mRNA stability, and translational repressors or activators
• mRNA degradation pathways that control the turnover and stability of mRNA molecules in the cell
  • Deadenylation shortening of the poly(A) tail by deadenylase enzymes
  • Decapping removal of the 5' cap by decapping enzymes, exposing the mRNA to 5' to 3' exonucleolytic degradation
• Nonsense-mediated decay (NMD) pathway that targets mRNAs with premature stop codons for degradation to prevent the production of truncated or non-functional proteins
• RNA interference (RNAi) mechanism of gene silencing mediated by small non-coding RNAs (siRNAs, miRNAs) that target complementary mRNAs for degradation or translational repression

Applications and Relevance in Cell Biology

• Gene expression profiling analysis of mRNA levels to study cell-type-specific gene expression, developmental processes, and cellular responses to stimuli
• Protein engineering modification of protein sequences to alter their properties (stability, specificity, activity) for biotechnological and therapeutic applications
• Recombinant protein production synthesis of proteins in host cells (bacteria, yeast, mammalian cells) for research, industrial, and medical purposes
• Genetic disorders caused by mutations that affect protein synthesis or function (sickle cell anemia, cystic fibrosis, Huntington's disease)
• Targeted therapies drugs or molecules designed to specifically interfere with disease-related proteins or their synthesis (small molecule inhibitors, antisense oligonucleotides, RNA interference)
• Synthetic biology design and construction of artificial biological systems or organisms with novel functions by manipulating genetic circuits and protein synthesis pathways
• Evolutionary studies analysis of protein sequences and structures across species to infer evolutionary relationships and functional conservation
• Biomarkers proteins or mRNAs used as indicators of physiological states, disease progression, or response to treatment in diagnostic and prognostic applications