Download Incomplete handout (Lecture 2) - the Conway Group

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 •
•
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
Dr Stuart Conway
Organic Option II: Chemical Biology
University of Oxford
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
Dr Stuart Conway
Organic Option II: Chemical Biology
University of Oxford
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 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. 4
Dr Stuart Conway
Organic Option II: Chemical Biology
University of Oxford
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 •
•
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. slide 47 5
Dr Stuart Conway
Organic Option II: Chemical Biology
University of Oxford
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. 6
Dr Stuart Conway
Organic Option II: Chemical Biology
University of Oxford
The genetic code •
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. slide 51 •
•
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
Dr Stuart Conway
Organic Option II: Chemical Biology
University of Oxford
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. •
•
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. •
Translation •
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. slide 53 8
Dr Stuart Conway
Organic Option II: Chemical Biology
University of Oxford
Modified nucleotides that occur in tRNA slide 54 The structure of yeast tRNAPhe slide 55 9
Dr Stuart Conway
Organic Option II: Chemical Biology
University of Oxford
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
Dr Stuart Conway
Organic Option II: Chemical Biology
University of Oxford
Translation slide 62 •
The ribosomal peptidyl transfer reaction occurs ~107-­‐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
Dr Stuart Conway
Organic Option II: Chemical Biology
University of Oxford
Translation slide 64 •
14