🧬Molecular Biology Unit 6 – Translation and Protein Synthesis

Key Concepts and Terminology

• Translation converts the genetic information in mRNA into a polypeptide chain
• Transcription produces mRNA from a DNA template in the nucleus
• Codons are triplets of nucleotides that specify amino acids or stop signals
• Anticodons are complementary triplets found on tRNA molecules
• Ribosomes are the sites of protein synthesis and consist of rRNA and proteins
  • Small ribosomal subunit binds to mRNA and mediates the binding of tRNA
  • Large ribosomal subunit catalyzes the formation of peptide bonds
• Aminoacyl-tRNA synthetases attach specific amino acids to their corresponding tRNAs
• Polyribosomes (polysomes) are multiple ribosomes translating the same mRNA simultaneously

DNA to RNA: Transcription Process

• RNA polymerase binds to the promoter region of a gene and separates the DNA strands
• Transcription factors help recruit RNA polymerase and initiate transcription
• Elongation occurs as RNA polymerase moves along the DNA template strand in the 5' to 3' direction
  • Nucleotides are added to the growing RNA strand complementary to the template strand
  • RNA sugar-phosphate backbone forms via phosphodiester bonds
• Termination occurs when RNA polymerase reaches a termination signal (sequence)
  • Transcription factors and termination factors help release the RNA polymerase and newly synthesized RNA
• Primary transcript (pre-mRNA) undergoes processing before leaving the nucleus as mature mRNA

mRNA Processing and Modification

• 5' cap addition involves adding a 7-methylguanosine to the 5' end of the pre-mRNA
  • Protects mRNA from degradation and facilitates ribosome binding during translation
• 3' polyadenylation adds a poly(A) tail (150-250 adenine residues) to the 3' end of the pre-mRNA
  • Enhances mRNA stability and aids in export from the nucleus
• Splicing removes introns (non-coding regions) and joins exons (coding regions) together
  • Spliceosome (complex of snRNPs and proteins) catalyzes the splicing reaction
• Alternative splicing allows for the production of different mRNA variants from the same pre-mRNA
  • Contributes to protein diversity and gene regulation
• Mature mRNA is exported from the nucleus to the cytoplasm for translation

The Genetic Code

• Genetic code is the set of rules that defines the relationship between codons and amino acids
• 64 possible codons: 61 specify amino acids, and 3 are stop codons (UAA, UAG, UGA)
• Degenerate code multiple codons can code for the same amino acid
• Unambiguous each codon specifies only one amino acid or stop signal
• Universality genetic code is nearly identical across all living organisms
  • Exceptions in mitochondrial and some protist genetic codes
• Start codon (AUG) initiates translation and codes for methionine
• Stop codons (UAA, UAG, UGA) terminate translation and do not code for amino acids

tRNA and Amino Acids

• tRNAs are adapter molecules that link codons in mRNA to amino acids
• Cloverleaf secondary structure of tRNA with four main arms (acceptor, D, anticodon, and TΨC)
• Anticodon loop contains the anticodon that base-pairs with the corresponding codon in mRNA
• Acceptor stem contains the 3' CCA end, where the amino acid is attached
• Aminoacyl-tRNA synthetases (aaRS) attach specific amino acids to their cognate tRNAs
  • Two-step reaction: activation of amino acid with ATP and transfer to tRNA
• Proofreading mechanisms ensure accurate aminoacylation and prevent errors in protein synthesis

Ribosome Structure and Function

• Ribosomes are ribonucleoprotein complexes that catalyze protein synthesis
• Consist of two subunits: small (30S in prokaryotes, 40S in eukaryotes) and large (50S in prokaryotes, 60S in eukaryotes)
• Small subunit binds mRNA and facilitates the binding of tRNA anticodons to mRNA codons
• Large subunit contains the peptidyl transferase center (PTC) that catalyzes peptide bond formation
• Three tRNA binding sites: A (aminoacyl), P (peptidyl), and E (exit)
  • tRNAs move sequentially from the A site to the P site and then to the E site during translation
• Ribosomes can associate with the endoplasmic reticulum (ER) to synthesize membrane and secretory proteins

Steps of Translation

• Initiation begins with the assembly of the initiation complex
  • Small ribosomal subunit binds to the 5' cap of mRNA and scans for the start codon (AUG)
  • Initiation factors (IFs) and initiator tRNA (Met-tRNAi) help form the initiation complex
• Elongation occurs as the ribosome moves along the mRNA, adding amino acids to the growing polypeptide chain
  • Aminoacyl-tRNA enters the A site, and its anticodon base-pairs with the mRNA codon
  • Peptidyl transferase catalyzes the formation of a peptide bond between the new amino acid and the growing polypeptide chain
  • Ribosome translocates to the next codon, moving the tRNAs from A to P and P to E sites
• Termination happens when the ribosome encounters a stop codon (UAA, UAG, or UGA)
  • Release factors (RFs) recognize the stop codon and trigger the hydrolysis of the peptidyl-tRNA bond
  • Polypeptide chain is released, and the ribosomal subunits dissociate from the mRNA

Protein Folding and Modifications

• Newly synthesized polypeptide chains fold into their native three-dimensional structures
• Primary structure the linear sequence of amino acids in a polypeptide chain
• Secondary structure regular local folding patterns (α-helices and β-sheets) stabilized by hydrogen bonds
• Tertiary structure overall three-dimensional shape of a polypeptide chain
  • Stabilized by interactions between amino acid side chains (disulfide bonds, hydrophobic interactions, etc.)
• Quaternary structure association of multiple polypeptide subunits in a multi-subunit protein
• Chaperones assist in proper protein folding and prevent aggregation
• Post-translational modifications (PTMs) covalent changes to proteins after translation
  • Examples: phosphorylation, glycosylation, acetylation, and ubiquitination
  • PTMs can affect protein function, stability, localization, and interactions

Regulation of Protein Synthesis

• Transcriptional control regulates the synthesis of mRNA from DNA
  • Transcription factors bind to regulatory sequences (promoters, enhancers) to control gene expression
• Translational control regulates the synthesis of proteins from mRNA
  • Modulation of initiation factors, RNA-binding proteins, and microRNAs can affect translation efficiency
• mRNA stability and degradation rates influence protein levels
  • Cis-acting elements (AU-rich elements) and trans-acting factors (RNA-binding proteins) regulate mRNA stability
• Protein degradation balances protein synthesis to maintain cellular homeostasis
  • Ubiquitin-proteasome system selectively degrades proteins tagged with ubiquitin
  • Autophagy degrades cytoplasmic components, including proteins and organelles

Applications and Relevance

• Understanding protein synthesis is crucial for developing treatments for genetic diseases caused by mutations in genes
• Recombinant DNA technology allows for the production of proteins (insulin, growth hormones) in host cells
• Studying translation in viruses can lead to the development of antiviral drugs that target viral protein synthesis
• Investigating the regulation of protein synthesis in cancer cells may help identify new therapeutic targets
• Manipulating protein synthesis pathways in plants can improve crop yield, nutritional content, and stress resistance
• Protein engineering techniques (site-directed mutagenesis) can create proteins with novel or enhanced functions
• Studying the evolution of the genetic code and translation machinery provides insights into the origin of life