Organic Chemistry Option II: Chemical Biology Recommended books: Dr Stuart Conway Department of Chemistry, Chemistry Research Laboratory, University of Oxford email: stuart.conway@chem.ox.ac.uk Teaching webpage (to download hand- outs): http://conway.chem.ox.ac.uk/teaching.html Biochemistry 4 th 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
RNA synthesis: Transcription slide 39 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 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. The DNA- RNA hybrid helix consists of antiparallel strands, hence the DNA s template strand is read in its 3 5 direction. 2
RNA polymerase slide 41 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 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. 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. 3
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. A doublet code would only allow 4 2 = 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. 4
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. slide 46 Or is the code non- overlapping? E.g. ABC specifies the first amino acid and DEF the second etc. slide 47 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. 5
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. 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. 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. 6
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). 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. 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. 7
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. 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 14 C labelled amino acids become bound to low molecular mass fractions of RNA. 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. 8
Modified nucleotides that occur in trna slide 54 The structure of yeast trna Phe slide 55 9
Synthesis of trna slide 56 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. Elucidating the molecular structure of the ribosome has been extremely challenging. 10
Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford Ribosome slide 59 11
Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford Translation slide 60 Translation slide 61 12
Translation slide 62 The ribosomal peptidyl transfer reaction occurs ~10 7 - fold faster than the uncatalysed reaction. Translation slide 63 The ribosome may also play a role in excluding water from the preorganised electrostatic environment of the active site. 13
Translation slide 64 14