Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford
Organic Chemistry Option II: Chemical Biology
Dr Stuart Conway Department of Chemistry, Chemistry Research Laboratory, University of Oxford email: [email protected] Teaching webpage (to download hand-outs): http://conway.chem.ox.ac.uk/Teaching.html
Recommended books:
Biochemistry 4th Edition by Voet and Voet, published by Wiley, ISBN: 978-0-470-57095-1.
Foundations of Chemical Biology by Dobson, Gerrard and Pratt, published by OUP (primer) ISBN: 0-19-924899-0
1 Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford
RNA synthesis: Transcription slide 39
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• It catalyses the DNA-directed coupling of nucleotide triphosphates to synthesise new RNA.
• The newly synthesised RNA is complementary to the template DNA.
Transcription slide 40
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• Hence, the incoming nucleotide is added to the free 3’-OH of the growing RNA chain.
• RNA polymerase selects the nucleotide it incorporates into the growing RNA chain based on the requirement that it forms a Watson-Crick base pair with the DNA strand that is being transcribed (the template strand - only one strand of DNA is transcribed at a time).
• The RNA polymerase moves along the DNA duplex that it is transcribing and separates a short (~14 base pairs) segment of the DNA helix to form a transcription bubble.
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• The DNA-RNA hybrid helix consists of antiparallel strands, hence the DNA’s template strand is read in its 3’→5’ direction.
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RNA polymerase slide 41
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• The outer surface of the protein is almost uniformly negatively charges, whereas the surfaces that interact with nucleic acids are positively charged.
• The DNA occupies the main channel, which directs the template strand to the active site.
• There the DNA base-pairs with the incoming nucleotide triphosphate (not in structure).
Translation slide 42
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• Although the formation of a peptide bond is relatively simple, the translational process in highly complicated.
• This complexity arises from the need to link 20 different amino acids residues accurately in the order specified by a particular mRNA.
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• As the base sequence of DNA is the only variable element in this otherwise monotonously repeating polymer, the base sequence and the protein sequence must be linked.
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Translation slide 43
“The problem of how a sequence of four things can determine a sequence of twenty things is known as the coding problem.”
Translation slide 44
• With only 4 bases in DNA to code for 20 amino acids, a group of several bases (a codon) is necessary to specify a single amino acid.
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• A doublet code would only allow 42 = 16 codons, which is insufficient to specify 20 amino acids.
• In a triplet code as many as 44 codons might not code for amino acids.
• Alternatively, some amino acids might be specified by more than one codon - a degenerate code.
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The genetic code slide 45
• How is DNA’s continuous sequence grouped into codons?
• Is the code overlapping? E.g. ABC codes for the first amino acids and BDC codes for the second etc.
The genetic code slide 46
• Or is the code non-overlapping?
• E.g. ABC specifies the first amino acid and DEF the second etc.
The genetic code slide 47
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• The genetic code is highly degenerate: Three amino acids (L, R, S) are each specified by six codons.
• Only Met and Trp, two of the least common amino acids in proteins, are specified by a single codon.
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The genetic code slide 48
• Sydney Brenner and Francis Crick formed the following hypotheses on the genetic code:
1. The code is a triplet code.
2. The code is read in a sequential manner starting from a fixed point in the gene. The insertion or deletion of a nucleotide shifts the frame (grouping) in which in which the succeeding nucleotides are read as codons. Thus the code has no internal punctuation that indicates the reading frame - the code is comma free.
3.
The genetic code slide 49
• The sentence represents a gene in which the words (codons) each contain three letters (bases).
• The spaces have no physical significance; they only present to indicate the reading frame.
• The deletion of the fourth letter (B) shifts the reading frame so that all of the words after the deletion are meaningless - specify the wrong amino acids.
The genetic code slide 50
• Insertion of a letter (X) passed the point of the original mutation restores the original reading frame.
• Hence on the words (codons) between the two changes (mutations) are altered.
• Therefore the sentence may still be intelligible (the gene could still specify a functional protein), particularly if the changes are close together.
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The genetic code slide 51
• The major breakthrough in deciphering the genetic code came in 1961 when Nirenberg and Matthaei established that UUU is the codon specifying Phe.
• They added poly(U) to a cell-free protein synthesising system and showed that this stimulated synthesis of only poly(Phe).
• In similar experiments, poly(A) was shown to specify poly(Lys) and poly(C) was found to specify poly(Pro).
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• These stop codons are also known (somewhat inappropriately) as nonsense codons as they are the only codons that do not specify amino acids.
• UAG, UAA and UGA are sometimes referred to as ambre, ochre and opal codons.
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• These codons also specify amino acids, Met and Val, respectively.
• The arrangement of the genetic code is not random.
• Most synonyms (codons that only differ in their third nucleotide) occupy the same box in the table.
• XYU and XYC always specify the same amino acids; XYA and XYG do so in all by two cases.
• Changes in the first codon position tend to specify the same or similar amino acids.
• Codons with second position pyrimidines (C AND U) tend to specify hydrophobic amino acids.
• Codons with second position purines (A and G) encode mostly polar amino acids.
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The genetic code slide 52
• How does the information in DNA actually translate into polypeptide sequences?
• In 1955 Francis Crick proposed the adaptor hypothesis stating that translation occurs through the mediation of adaptor molecules.
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• Each adaptor was postulated to carry a specific amino acid and to recognise the corresponding codon.
• At a similar time it was shown that in the course of protein synthesis 14C labelled amino acids become bound to low molecular mass fractions of RNA.
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Translation slide 53
• All tRNAs contain:
• A 5’-terminal phosphate.
• A 7-base pair step that includes the 5’- terminal nucleotide and may include non-Watson-Crick base pairs, such as G ⋅ U. This assembly is known as the acceptor stem as the amino acid is appended to the 3’-OH group.
• A 3- or 4-base stem ending in a loop that that frequently contains the modified base dihydrouridine (D), known as the D arm.
• A 5-base-pair stem ending in a loop that usually contains the sequence TΨC (Ψ = pseudouridine).
• All tRNAs terminate in the sequence CCA, with a free 3’-OH group.
• There are 15 invariant positions and 8 semi-invariant (only a purine or only a pyrimidine) positions.
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Modified nucleotides that occur in tRNA slide 54
Phe The structure of yeast tRNA slide 55
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Synthesis of tRNA slide 56
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• This mixed anhydride then reacts with tRNA to form aminoacyl-tRNA and AMP.
Ribosome slide 57
• For translation to occur, mRNA and tRNA must bind to each other, and the amino acids carried by the tRNA must react to form the polypetide chain.
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• Elucidating the molecular structure of the ribosome has been extremely challenging.
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Ribosome slide 59
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Translation slide 60
Translation slide 61
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Translation slide 62
• The ribosomal peptidyl transfer reaction occurs ~107-fold faster than the uncatalysed reaction.
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Translation slide 63
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• The ribosome may also play a role in excluding water from the preorganised electrostatic environment of the active site.
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Translation slide 64
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