1. PROTEINS
Nicky Mulder
Acknowledgements: Anna Kramvis for lecture material (adapted here)

2. Central dogma of molecular biology 2

3. Protein building blocks
Proteins are made up of amino acids
20 possible amino acids
Each specified/encoded by a triplet of bases
Messenger RNA transcripts translated into proteins

4. The Genetic Code
Each amino acid is specified by a triplet of 3 bases (codons)
Codons were elucidated a decade after the discovery of the DNA structure in 1953
If we have the 4 bases A,C,G,T we have 4 x4 x4 = 64 possible codons
Actually 61 codons + 3 stop codons 4

5. The Genetic Code
Codon usage varies 5

6. Open reading frame
String of in-frame combinations/triplets of bases that specify an amino acid
Starts with ATG (Meth) or Val
Ends with stop codon
One base insertion or deletion –out of frame/frameshift

7. Translating sequences
6 possible reading frames, 3 in each direction
AGTCGGCTGACTGCGTTTACGAATGCGATTACTCCCTT +1
Reverse complement
AAGGGAGTAATCGCATTCGTAAACGCAGTCAGCCGACT -1

8. Translating sequences
6 possible reading frames, 3 in each direction
AGTCGGCTGACTGCGTTTACGAATGCGATTACTCCCTT +2
AAGGGAGTAATCGCATTCGTAAACGCAGTCAGCCGACT -2

9. Translating sequences
6 possible reading frames, 3 in each direction
AGTCGGCTGACTGCGTTTACGAATGCGATTACTCCCTT +3
AAGGGAGTAATCGCATTCGTAAACGCAGTCAGCCGACT -3

10. Getting the final protein
Six-frame translation
Find longest ORF with initiation site, start codon and ending with stop codon

11. Transcription and translation
ATGCGGTGCAACGTGCATCCTAAA
UACGCCACGUUGCACGUAGGAUUU
W G P Y T A K L

12. http://www. virtualsciencefair. org/2004/mcgo4s0/public_html/t3/RNA 12

13. library.thinkquest.org

14. Ribosomes Protein synthesizers
Different subunits for interacting with mRNA and tRNAs 14

15. Translation process
Copyright-Anna Kramvis 15 15

16. Amino acid structure
The chemistry of R groups distinguishes amino acids and their properties

17. Polypeptide chain
Each protein has a unique sequence of amino acids joined into a polypeptide chain
Already in 1905 it was known that proteins consist of amino acids linked by peptide bonds. Chain lengths vary from 5 to 4000 amino acids.
There are 20 amino acids and proteins consist of various mixtures of these amino acids.
Valine Leucine Serine Tyrosine Proline 17

18. Protein primary structure
Proteins made up amino acids joined by peptide bonds between carboxyl group of one and amino group of the next
commons.wikimedia.org

19. Peptide backbone

20. Primary structure, disulphide bonds

21. Secondary structure
Held together by interactions (H-bonds) between peptide backbones

22. Tertiary structure
Tertiary structure is controlled by the interactions between non-adjacent amino acid R groups

23. Quaternary Structure
More than one protein chain, e.g. hemoglobin

24. Possible bonds in proteins
Hydrogen bonds: weak electrostatic attractions between electronegative atom (O or N).
Van der Waals forces: can be attractive or repulsive, depends on distance
Electrostatic interactions or ionic bonds: weak bonds that form between charged groups in aqueous environments
Hydrophobic effects: arise because hydrogen bonded structure of water forces hydrophobic groups into the internal parts of the protein.

25. Other structures 25

26. Summary of protein structures

27. The function of a protein depends on sequence of amino acids and requires a precise folding of its polypeptide chain 27

28. Properties of Amino Acids 28

29. Hydrophobic, two chiral carbons Proline P Pro
Name
R-Group Properties
Glycine G
Gly
Hydrophobic
Alanine A
Ala
Valine V
Val
Leucine L
Leu
Isoleucine I
Ile
Hydrophobic, two chiral carbons
Proline P
Pro
Cyclic, not terribly hydrophobic
Phenylalanine F
Phe
Hydrophobic, bulky
Tyrosine Y
Tyr
Less hydrophobic (than Phe), bulky
Tryptophan W
Trp
Hydrophobic, bulky (indole ring)
Cysteine C
Cys
Hydrophobic, highly reactive (S-S link)
Methionine M
Met
Hydrophobic (start a.a.)
Serine S
Ser
Hydrophilic, reactive
Threonine T
Thr
Hydrophilic, reactive, two chiral carbons
Lysine K
Lys
Highly hydrophilic, positively charged
Arginine R
Arg
Histidine H
His
Highly hydrophilic, positive or neutral
Aspartate D
Asp
Highly hydrophilic, negatively charged
Glutamate E
Glu
Asparagine N
Asn
Uncharged
Glutamine Q
Gln
Copyright-Anna Kramvis 29 29

30. Some protein functions
proteins
enzymes
bind ions
structural
membranes
connective tissue
muscle fibres
extracellular
hormones
antibodies 30

31. Information from a protein sequence
MDITIQHPWFKRALGSLYPSRLFDQFFGEGLFEYDLLPFLSSTISPYYRQSLFR
amino acid composition
molecular weight

32. Information from a protein sequence
Single amino acid physical properties
MDQHPWFKRAITIVLLGLLPFLSLYPSRLFDQFCGEGLFEYDSSTISCYRQSLFRTVLESG
D,E -acidic
C,D,E,H,K,N,Q,R,S,T –polar, active sites, metal binding
V,L,I,M –hydrophobic, membrane
C –disulphide-rich, disulphide bonds

33. Information from a protein sequence
Functionally important regions
MDQHPWFKRAITIVLLGLLPFLSLYCPSRLFDQFCGEGLFEYDSSTISYRQSLFRTNVLES
Active site/metal binding
Hydrophobic region
Glycosylation site
disulphide bond
Transmembrane regions
Signal sequences
Localisation signals (subcellular location)
Targeting sequences
Modification sites

34. Information from a protein sequence
FAMILY
Conserved sequence
DOMAIN
Conserved domains
MOTIF
Properties of regions
SITE
RESIDUE
Physical amino acid properties
GKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYN

35. Protein abundance
Not all genes are expressed all the time, amount of protein is affected by:
gene expression -transcriptional regulation
Post-transcriptional regulation
Translational regulation
Post-translational regulation

36. Transcription regulation
Regulators –enhancers and repressors, can be cis- or trans-acting
Bind to specific sites
Sigma factors, anti-sigma factors
DNA unwinding
DNA methylation
Signalling pathways

37. Post-transcriptional regulation
mRNA half-life
Antisense RNA
RNA splicing
siRNAs

38. Translational regulation
Ribosomes
Translation factors
tRNA availability

39. Post-translational regulation
Transport to appropriate place
Protein folding (chaperones)
Post-translational modification:
Phosphorylation
Acetylation
Sugars added….

40. Summary of main building blocks of biological systems 40

41. Translation exercise Translate this mRNA using the genetic code table
5’AUGUUUUUGUCGUACUGGUGUCUACCUCAUCAACGUAUUACGAAUAAG3’
Write out the translation using the one letter and three letter conventions.
2. Give the characteristics of each amino acid in the polypeptide chain.
3. How long is the original RNA sequence and how long is the protein sequence?
Copyright-Anna Kramvis 41 41

42. Additional questions Here is a gene sequence:
5’ AGCAATGCATGCATCGTTATGG 3’
Identify the initiation codon
What reading frame is it in?
Would translation be affected if the first C was changed to G, if so, what effect?
Would translation be affected if the second last C was changed to T, if so, what effect?