🔬Biological Chemistry I Unit 14 – Protein Synthesis and Regulation

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 three-nucleotide sequences in mRNA that code for specific amino acids or stop signals during translation
  • Start codon

    AUG

    initiates translation and codes for methionine
  • Stop codons (

    UAA

    ,

    UAG

    ,

    UGA

    ) terminate translation
• Anticodons are complementary three-nucleotide sequences found on tRNA molecules that base pair with codons in mRNA
• Ribosomes are complex molecular machines composed of rRNA and proteins that catalyze the synthesis of polypeptide chains during translation
• Promoters are DNA sequences upstream of genes that recruit RNA polymerase and transcription factors to initiate transcription
• Enhancers are distal regulatory elements that can increase gene expression by interacting with promoters and transcription factors

DNA Structure and Function

• DNA is a double-stranded helical molecule composed of nucleotides held together by hydrogen bonds between complementary base pairs (A-T and G-C)
• The sugar-phosphate backbone of DNA is on the outside of the helix, while the nitrogenous bases face the interior
• DNA stores genetic information in the sequence of its nucleotides, which is used as a template for RNA synthesis and ultimately protein synthesis
• The double helix structure of DNA provides stability and protection against degradation while allowing for efficient replication and transcription
• DNA is organized into chromosomes in eukaryotic cells, with each chromosome containing numerous genes and regulatory elements
• Chromatin is the complex of DNA and proteins (histones) that helps package DNA into a compact structure within the nucleus
  • Euchromatin is loosely packed and transcriptionally active, while heterochromatin is tightly packed and transcriptionally silent

Transcription Process

• Transcription is the synthesis of RNA from a DNA template catalyzed by RNA polymerase
• Transcription occurs in three stages: initiation, elongation, and termination
• During initiation, RNA polymerase binds to the promoter region of a gene with the help of transcription factors, forming the transcription initiation complex
• In elongation, RNA polymerase moves along the DNA template strand in the 5' to 3' direction, synthesizing a complementary RNA strand by adding nucleotides to the growing 3' end
• Termination occurs when RNA polymerase encounters a termination signal, causing the release of the newly synthesized RNA and dissociation of the polymerase from the DNA template
• In eukaryotes, transcription occurs in the nucleus and is carried out by three different RNA polymerases (I, II, and III), each responsible for transcribing specific classes of RNA
  • RNA polymerase I transcribes ribosomal RNA (rRNA)
  • RNA polymerase II transcribes messenger RNA (mRNA) and some small nuclear RNAs (snRNAs)
  • RNA polymerase III transcribes transfer RNA (tRNA) and other small RNAs

RNA Processing and Modification

• Eukaryotic RNA undergoes several processing and modification steps before becoming functional
• Pre-mRNA splicing removes non-coding introns and joins coding exons to form mature mRNA
  • Splicing is catalyzed by the spliceosome, a complex of snRNAs and proteins
  • Alternative splicing allows for the production of multiple mRNA isoforms from a single gene, increasing protein diversity
• 5' capping adds a 7-methylguanosine cap to the 5' end of the mRNA, which aids in translation initiation and protects the mRNA from degradation
• 3' polyadenylation adds a poly(A) tail to the 3' end of the mRNA, which enhances stability and facilitates export from the nucleus
• RNA editing modifies specific nucleotides in the RNA sequence, such as the conversion of adenosine to inosine (A-to-I editing) or cytidine to uridine (C-to-U editing)
• tRNA molecules undergo extensive modifications, including the addition of the CCA sequence at the 3' end, which is essential for amino acid attachment
• rRNA is processed and modified to form the functional components of the ribosome, including the small (40S) and large (60S) subunits in eukaryotes

Translation Mechanism

• Translation is the process of synthesizing a polypeptide chain from an mRNA template, carried out by ribosomes
• Translation occurs in three stages: initiation, elongation, and termination
• Initiation involves the assembly of the translation initiation complex, which includes the small ribosomal subunit, initiator tRNA (Met-tRNAi), and initiation factors
  • The initiation complex recognizes the start codon (AUG) on the mRNA and positions the Met-tRNAi in the P site of the ribosome
• During elongation, the ribosome moves along the mRNA in the 5' to 3' direction, adding amino acids to the growing polypeptide chain
  • Aminoacyl-tRNA synthetases attach specific amino acids to their cognate tRNAs, forming aminoacyl-tRNAs
  • The anticodon of the aminoacyl-tRNA base pairs with the corresponding codon on the mRNA in the A site of the ribosome
  • The peptidyl transferase center of the ribosome catalyzes the formation of a peptide bond between the amino acid on the A site tRNA and the growing polypeptide chain on the P site tRNA
  • Translocation moves the ribosome one codon downstream, shifting the tRNAs from the A and P sites to the P and E sites, respectively
• Termination occurs when the ribosome encounters a stop codon (UAA, UAG, or UGA) on the mRNA
  • Release factors recognize the stop codon and trigger the hydrolysis of the peptidyl-tRNA bond, releasing the completed polypeptide chain
  • The ribosomal subunits, mRNA, and tRNAs dissociate, ready for another round of translation

Protein Structure and Folding

• Proteins are linear polymers of amino acids that fold into specific three-dimensional structures to perform their biological functions
• The primary structure of a protein is the linear sequence of amino acids, determined by the genetic code
• Secondary structure refers to the local folding patterns of the polypeptide chain, such as α-helices and β-sheets, stabilized by hydrogen bonds
• Tertiary structure is the overall three-dimensional shape of a single polypeptide chain, resulting from interactions between secondary structure elements and side chains
  • Tertiary structure is stabilized by various non-covalent interactions, such as hydrogen bonds, van der Waals forces, and hydrophobic interactions, as well as covalent disulfide bonds
• Quaternary structure is the arrangement of multiple polypeptide chains (subunits) in a multi-subunit protein complex
• Protein folding is the process by which a polypeptide chain acquires its native three-dimensional structure
  • Chaperones are proteins that assist in the folding process by preventing aggregation and promoting proper folding
  • Misfolded proteins can lead to various diseases, such as Alzheimer's and Parkinson's, characterized by the accumulation of protein aggregates

Regulation of Gene Expression

• Gene expression is tightly regulated to ensure that the right proteins are produced at the right time and in the right amounts
• Transcriptional regulation controls the initiation and rate of transcription through the interaction of transcription factors with promoters and enhancers
  • Transcriptional activators bind to enhancers and promote the assembly of the transcription initiation complex
  • Transcriptional repressors bind to silencers and inhibit transcription by blocking the binding of activators or RNA polymerase
• Epigenetic modifications, such as DNA methylation and histone modifications, can alter chromatin structure and influence gene expression without changing the DNA sequence
  • DNA methylation typically occurs at CpG dinucleotides and is associated with gene silencing
  • Histone acetylation is generally associated with active transcription, while histone deacetylation is associated with repression
• Post-transcriptional regulation controls the processing, stability, and translation of mRNA
  • RNA-binding proteins (RBPs) can influence mRNA splicing, stability, and localization
  • MicroRNAs (miRNAs) are small non-coding RNAs that can repress translation or promote mRNA degradation by base pairing with complementary sequences in the target mRNA
• Translational regulation controls the rate and efficiency of protein synthesis
  • Translation initiation can be regulated by the availability of initiation factors and the accessibility of the start codon
  • Upstream open reading frames (uORFs) in the 5' untranslated region (UTR) of mRNA can modulate translation efficiency
• Feedback inhibition is a common mechanism for regulating gene expression, where the end product of a pathway inhibits the activity of an earlier enzyme in the pathway

Post-Translational Modifications

• Post-translational modifications (PTMs) are covalent modifications of proteins that occur after translation and can influence protein function, stability, and localization
• Phosphorylation is the addition of a phosphate group to serine, threonine, or tyrosine residues by protein kinases
  • Phosphorylation can activate or inactivate enzymes, alter protein-protein interactions, and create binding sites for other proteins
  • Dephosphorylation by protein phosphatases reverses the effects of phosphorylation
• Glycosylation is the attachment of carbohydrate moieties to proteins, which can occur on asparagine (N-linked) or serine/threonine (O-linked) residues
  • Glycosylation can influence protein folding, stability, and interactions with other molecules
• Ubiquitination is the covalent attachment of ubiquitin, a small protein, to lysine residues of target proteins
  • Polyubiquitination often marks proteins for degradation by the proteasome
  • Monoubiquitination can regulate protein localization and interactions
• Acetylation is the addition of an acetyl group to lysine residues, catalyzed by acetyltransferases
  • Acetylation can neutralize the positive charge of lysine, affecting protein-protein interactions and DNA binding
  • Deacetylation by deacetylases reverses the effects of acetylation
• Methylation is the addition of one or more methyl groups to lysine or arginine residues, catalyzed by methyltransferases
  • Methylation can influence protein-protein interactions and gene expression, particularly in the context of histone modifications

Applications and Real-World Examples

• Recombinant DNA technology allows for the production of human proteins in bacterial or eukaryotic expression systems for therapeutic use (insulin, growth hormone)
• Gene therapy involves the introduction of functional genes into cells to replace defective or missing genes, potentially treating genetic disorders (sickle cell anemia, cystic fibrosis)
• CRISPR-Cas9 is a powerful genome editing tool that can be used to make precise changes to DNA sequences, with applications in basic research, agriculture, and medicine (disease modeling, gene correction)
• RNA interference (RNAi) is a natural process that can be harnessed to silence specific genes using small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), with potential therapeutic applications (cancer, viral infections)
• Personalized medicine utilizes an individual's genetic information to tailor medical treatments and preventive strategies (pharmacogenomics, cancer genomics)
• Synthetic biology combines principles of biology and engineering to design and construct novel biological systems or organisms with desired functions (biofuels, biosensors, drug delivery)
• Protein engineering involves the rational design or directed evolution of proteins to improve their stability, specificity, or catalytic activity for various applications (industrial enzymes, antibody therapeutics)
• Studying the regulation of gene expression and protein function in model organisms (mice, fruit flies, zebrafish) provides insights into human development, physiology, and disease