MBG 2040 Lecture Notes - Lecture 9: Van Der Waals Force, Aminoacyl-Trna, Neurospora Crassa
2 May 2018
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Translation and the Genetic Code
Sickle Cell Anemia: caused by a single amino acid change (Glu --> Val) in one of the protein changing
that make up hemoglobin in red blood cells
• Hemoglobin is created in non-functional form
• Can no longer efficiently transport and provide oxygen
• Usually tends to be fatal if not treated
Lecture Outline:
• Protein structure and amino acids
• Protein synthesis: translation
• The genetic code
• Codon-tRNA interactions
Overview of Gene Expression: the central dogma
• DNA --> mRNA via transcription (RNA synthesis)
• mRNA --> protein via translation (protein synthesis)
o Mediated by tRNAs and ribosome
Amino Acids:
• Proteins are made of single or multiple polypeptides
• A polypeptide is a long chain of amino acid residues comprising 20 different kinds of amino acids
• Amino acids have three parts:
o Free amino group
o Free carboxyl group
o Side group (R)
*see slide
• Amino acids can be classified by it's side group:
o Hydrophobic or non-polar (-H, -CH3)
o Hydrophilic or polar (-OH, =O, -SH)
o Acidic (-COOH)
o Basic (-H2N, =NH, -NCH2)
• Peptide Bonds:
o Amino acid residues are joined by peptide bonds
o The carboxyl group of one amino acid is covalently linked to the amino group of the next
amino acid
o Polypeptide chain goes from the N to the C terminus
o *see slide
Four Levels of Protein Structure (Hemoglobin)
• Primary - chain of amino acids
• Secondary - fold of amino acid chain; formation of secondary structures (alpha-helix, beta-sheets,
bonds)
• Tertiary - folding into specific 3D structure
o Beta-globin polypeptide with heme group
• Quaternary (functional protein) - multiple polypeptides
o Beta-globin polypeptides associate with alpha-globin polypeptides (and other heme
molecules)
*note: molecular interactions determine the tertiary structure of a polypeptide
• Hydrogen bond (OH--O)
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• Ionic bond (between opposite charges)
• Hydrophobic interaction (CH3 to CH3)
• Disulphide bridge (S-S)
• Van der Waals Interaction (CH2-OH to CH2-OH)
Summary:
• Most genes manifest their phenotypic effect(s) of an organism through proteins, which are large
macro-molecules composed of polypeptides
• Each polypeptide is a chain-like polymer made from different amino acids
• The amino acid sequence of a polypeptide is specified by the nucleotide sequence of a gene (its
mRNA)
• The vast functional diversity of proteins results in part from their complex 3D structures
From 'One Gene, One Enzyme' to 'One Gene, One Polypeptide': evolution of the concept of a gene
• Sequence of nucleotides in a gene specifies a co-linear sequence of amino acids in its polypeptide
product
• 'One gene one enzyme' hypothesis:
o George Beadle and Edward Tatum: Nobel Prize in 1958
• Step 1: wild-type spores (on minimal medium) are irradiated, and the resulting strains
are crossed with wild-type
▪ X-rays or ultraviolet are applied to conidia (asexual spores are haploid but
nucleate)
▪ Mycelium are grown from a single irradiated spore
▪ Spore becomes a fruiting body via meiosis
▪ --> sexual spore (haploid and uninucleate)
▪ Transferred to a complete medium (with vitamins, amino acids…etc.)
• Step 2: individual ascospores are tested for general growth requirements
▪ Mycelium only grew on complete medium or on medium with vitamins added
▪ Note: different groups of nutrients are supplemented to the minimal medium
▪ Conclusion: vitamins are required for the fungal mutant to grow
• Radiation changed some sort of process
• Step 3: individual strains are tested for specific growth requirements
▪ Growth only occurred when vitamin pantothenic acid is added
• Co-linearity between a gene and its protein
o Prokaryotes:
• Ex. trpA gene and the alpha polypeptide of tryptophan synthetase (Charles Yanofsky)
• DNA (nucleotides) on genetic made produce amino acids in wild-type polypeptide in
linear form
• When mutated, each amino acid is changed
▪ Changes position of amino acid in polypeptide (still in order)
o Eukaryotes:
• Base-pair triplets in coding region: exon 1- intron 1 - exon 2
• Codons in primary transcript (via transcription) - including intron and exon
• Codons in mRNA (via processing of gene transcript) - intron gets spliced out
▪ Exons are conserved in a sequence
• Amino acids in polypeptide gene protein (via translation) - each amino acid
corresponds to each codon in exons
Summary:
• Beadle and Tatum's experiments with Neurospora crassa led to the 'one gene-one enzyme'
hypothesis, which was later modified to the 'one gene-one polypeptide' concept
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• The nucleotide sequences in a gene and amino acids in its polypeptide product are co-linear
Protein Synthesis: Translation
• Genetic information in an mRNA molecule is translated into the amino acid sequence of a
polypeptide according to the specifications of the genetic code
• Translation is carried out by the concerted actions of ribosome (rRNAs and proteins), tRNAs,
mRNAs, and soluble factors involved in three stages of translation
• Overview of Translation:
o DNA in chromosome
• rRNA precursor
▪ --> functional RNA molecules (5S, 16S, 23S, rRNAs)
• Combines with ribosomal proteins to create free ribosomal subunits
• mRNA
▪ --> translation: combines with ribosomal units and is influences by elongation
factors, transfer enzymes and GTP to produce a nascent peptide
• tRNA via RNA polymerase
▪ Create activating enzymes (aminoacyl~tRNAs)
• Macromolecules involved:
o Polypeptides (>50) and RNA molecules (304) that comprise the ribosome
o Amino-acid activating enzymes (amino-acyl tRNA synthetases; 20)
o tRNA molecules (at least 32)
o Soluble proteins involved in the initiation, elongation, and termination of a polypeptide chain
o mRNA to serve as the blueprint for translation
• Ribosomes:
o In Prokaryotes:
• 31 ribosomal proteins --> 5S rRNA --> 23S rRNA --> 50S subunit
• 21 ribosomal proteins --> 16S rRNA --> 30S subunit
• *these come together to form a 20nm 70S ribosome
o In Eukaryotes:
• 49 ribosomal proteins --> 5S rRNA --> 5.8S rRNA --> 28S rRNA --> 60S subunit
• 33 ribosomal proteins --> 18S rRNA --> 40S subunit
• *these come together to form 24nm 80S ribosome
• Synthesis and processing of the 30S precursor of rRNAs in E. coli
o rRNA gene on DNA is transcribed into a 30S precursor RNA
o Precursor rRNA is cleaved by endoribonuclease into individual precursors
• 16S rRNA, 4S tRNA, p23S rRNA, 5S rRNA
o As secondary cleavage by endoribonuclease and trimming by exoribonucleases produces
mature rRNAs
• Synthesis and processing of the 45S precursor of rRNAs in mammals
o rRNA gene in DNA is transcribed in larger primary transcript with 13,000 nucleotides
o Some regions are degraded and through RNA processing produces 18S rRNA, 5.8S rRNA,
and 28S rRNA
o *rRNA primer 3 --> 5S rRNA (exception)
• rRNA genes exist in multiple copies in both prokaryotes and eukaryotes
o rRNA genes in E. coli
• Seven rRNA genes are distributed among three locations on the circular chromosome
o rRNA genes in eukaryotes
• There are hundreds to thousands copies of rRNA genes
• The rRNA genes for 5.8S-18S-28S rRNAs are present in tandem arrays in the nucleolar
organizer regions of multiple chromosomes
• 5S rRNA genes are distributed over several chromosomes depending on the organism
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Document Summary
Lecture outline: protein structure and amino acids, protein synthesis: translation, the genetic code, codon-trna interactions. Overview of gene expression: the central dogma: dna --> mrna via transcription (rna synthesis, mrna --> protein via translation (protein synthesis, mediated by trnas and ribosome. *note: molecular interactions determine the tertiary structure of a polypeptide: hydrogen bond (oh--o) Ionic bond (between opposite charges: hydrophobic interaction (ch3 to ch3, disulphide bridge (s-s, van der waals interaction (ch2-oh to ch2-oh) From "one gene, one enzyme" to "one gene, one polypeptide": evolution of the concept of a gene: sequence of nucleotides in a gene specifies a co-linear sequence of amino acids in its polypeptide product. -> functional rna molecules (5s, 16s, 23s, rrnas: combines with ribosomal proteins to create free ribosomal subunits, mrna. In prokaryotes: 31 ribosomal proteins --> 5s rrna --> 23s rrna --> 50s subunit, 21 ribosomal proteins --> 16s rrna --> 30s subunit, *these come together to form a 20nm 70s ribosome.
