👨‍👩‍👦‍👦General Genetics Unit 9 – The Genetic Code and Translation

Key Concepts

• The genetic code is the set of rules that defines how the information encoded in genetic material (DNA or RNA sequences) is translated into proteins
• DNA stores genetic information, which is transcribed into RNA and then translated into proteins, the functional molecules of the cell
• The genetic code is nearly universal across all organisms, with a few rare exceptions (mitochondrial genetic code)
• Codons are three-nucleotide sequences in mRNA that specify amino acids or signal the start or stop of translation
  • 61 codons code for amino acids, while 3 codons serve as stop signals (UAA, UAG, UGA)
• The genetic code is degenerate, meaning multiple codons can code for the same amino acid (synonymous codons)
• Translation is the process of synthesizing a protein from an mRNA template, involving tRNAs, ribosomes, and various protein factors
• Mutations in the genetic code can lead to changes in the resulting protein sequence, potentially altering its function or causing genetic disorders

DNA and RNA Basics

• DNA (deoxyribonucleic acid) is a double-stranded molecule composed of nucleotides containing the bases adenine (A), thymine (T), guanine (G), and cytosine (C)
  • Complementary base pairing: A pairs with T, and G pairs with C
• RNA (ribonucleic acid) is a single-stranded molecule composed of nucleotides containing the bases adenine (A), uracil (U), guanine (G), and cytosine (C)
  • In RNA, uracil replaces thymine and pairs with adenine
• DNA is found primarily in the nucleus of eukaryotic cells and stores genetic information, while RNA is found in both the nucleus and cytoplasm and plays various roles in gene expression
• The central dogma of molecular biology describes the flow of genetic information: DNA is transcribed into RNA, which is then translated into proteins
• During transcription, the enzyme RNA polymerase synthesizes a complementary RNA strand from a DNA template
  • In eukaryotes, the primary transcript (pre-mRNA) undergoes processing (capping, splicing, polyadenylation) to form mature mRNA
• Messenger RNA (mRNA) carries the genetic information from DNA to the ribosomes for protein synthesis

The Genetic Code

• The genetic code is a triplet code, where each codon consists of three nucleotides that specify a particular amino acid or a stop signal
• The genetic code is read in a non-overlapping fashion, with codons read sequentially from a fixed starting point
• The genetic code is unambiguous, meaning each codon specifies only one amino acid or stop signal
• The start codon, AUG, codes for the amino acid methionine and initiates translation
  • In eukaryotes, the initial methionine is often removed post-translationally
• The genetic code exhibits wobble base pairing, where the third base of a codon can pair with multiple bases in the tRNA anticodon
  • This allows some tRNAs to recognize multiple codons for the same amino acid
• Mutations in the genetic code can be silent (no change in amino acid), missense (change in amino acid), or nonsense (premature stop codon)
• The redundancy of the genetic code provides some protection against the effects of mutations

tRNA and Ribosomes

• Transfer RNAs (tRNAs) are adapter molecules that bridge the genetic code and the amino acids they represent
  • Each tRNA has an anticodon complementary to a specific mRNA codon and carries the corresponding amino acid
• tRNAs have a cloverleaf secondary structure with four main arms: the acceptor arm, the D arm, the anticodon arm, and the TΨC arm
  • The acceptor arm carries the amino acid, while the anticodon arm contains the anticodon that base-pairs with the mRNA codon
• Aminoacyl-tRNA synthetases are enzymes that attach the correct amino acid to its corresponding tRNA in a two-step reaction
  • These enzymes ensure the fidelity of translation by correctly charging tRNAs with their cognate amino acids
• Ribosomes are the molecular machines that synthesize proteins by translating the genetic code in mRNA
  • Ribosomes consist of two subunits (large and small) composed of ribosomal RNA (rRNA) and proteins
• The ribosome has three tRNA binding sites: the A (aminoacyl) site, the P (peptidyl) site, and the E (exit) site
  • The A site binds incoming aminoacyl-tRNAs, the P site holds the growing polypeptide chain, and the E site releases uncharged tRNAs

Steps of Translation

• Translation occurs in three main stages: initiation, elongation, and termination
• Initiation:
  • In prokaryotes, the small ribosomal subunit binds to the Shine-Dalgarno sequence upstream of the start codon, while in eukaryotes, the small subunit recognizes the 5' cap and scans for the start codon
  • The initiator tRNA (carrying methionine) binds to the start codon in the P site, and the large ribosomal subunit joins to form the complete ribosome
• Elongation:
  • Aminoacyl-tRNAs enter the A site, and if the anticodon matches the mRNA codon, the ribosome catalyzes the formation of a peptide bond between the new amino acid and the growing polypeptide chain
  • The ribosome then translocates, moving the tRNAs and mRNA by one codon, such that the next codon is positioned in the A site
  • This process repeats, with the polypeptide chain growing one amino acid at a time
• Termination:
  • When a stop codon (UAA, UAG, or UGA) enters the A site, release factors bind and promote the hydrolysis of the peptidyl-tRNA bond
  • The completed polypeptide is released, and the ribosomal subunits dissociate from the mRNA and each other
• Post-translational modifications, such as folding, cleavage, and chemical modifications, may occur to produce the final functional protein

Protein Synthesis in Action

• Polyribosomes (polysomes) are clusters of ribosomes simultaneously translating the same mRNA, allowing for efficient protein synthesis
  • Multiple ribosomes can translate the same mRNA simultaneously, with each ribosome at a different stage of translation
• Co-translational protein folding occurs as the polypeptide chain emerges from the ribosome, with chaperone proteins assisting in the proper folding of the nascent protein
• Some proteins are synthesized as inactive precursors (preproteins) and require post-translational cleavage or modifications to become functional
  • Signal peptides direct proteins to specific cellular compartments (endoplasmic reticulum, mitochondria) and are cleaved after translocation
• The rate of protein synthesis can be regulated by factors such as mRNA stability, translational initiation factors, and the availability of charged tRNAs
• Antibiotics can target bacterial protein synthesis by inhibiting specific steps of translation (aminoacyl-tRNA binding, peptide bond formation, translocation)
  • Differences between prokaryotic and eukaryotic translation machinery allow for the development of antibiotics that selectively target bacterial protein synthesis

Regulation and Control

• Translation is regulated to control gene expression and respond to cellular needs and environmental cues
• Global control of translation occurs through the modification of translation factors or the availability of ribosomes
  • Phosphorylation of initiation factors can inhibit or promote translation initiation
• Specific control of translation can occur through regulatory elements in the mRNA, such as upstream open reading frames (uORFs) or internal ribosome entry sites (IRES)
  • uORFs can inhibit translation of the main ORF, while IRES allow for cap-independent translation initiation
• RNA-binding proteins (RBPs) can regulate translation by binding to specific sequences in the mRNA and promoting or inhibiting ribosome binding or progression
• 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
• Cellular stress conditions (heat shock, nutrient deprivation) can lead to a global reduction in translation and the formation of stress granules containing stalled translation initiation complexes
• Dysregulation of translation is associated with various diseases, including cancer, neurological disorders, and metabolic syndromes

Real-World Applications

• Understanding the genetic code and translation has enabled the development of recombinant DNA technology and the production of proteins in heterologous systems (bacteria, yeast, mammalian cells)
  • Insulin, growth hormones, and antibodies are examples of proteins produced using recombinant DNA technology
• Codon optimization involves altering the codons in a gene to match the codon usage bias of the host organism, improving protein expression levels
• Site-directed mutagenesis allows for the introduction of specific mutations in a gene, enabling the study of protein structure-function relationships and the creation of proteins with novel properties
• Nonsense suppression therapy aims to treat genetic diseases caused by premature stop codons by promoting the readthrough of these codons and the production of full-length proteins
• Ribosome profiling (Ribo-seq) is a technique that enables the genome-wide analysis of translation by sequencing ribosome-protected mRNA fragments, providing insights into translational regulation and codon usage
• Advances in cryo-electron microscopy have allowed for the high-resolution structure determination of ribosomes and other translation-related complexes, deepening our understanding of the molecular mechanisms of protein synthesis