proteins nicky mulder acknowledgements: anna kramvis for lecture material (adapted here)
TRANSCRIPT
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PROTEINS
Nicky Mulder
Acknowledgements: Anna Kramvis for lecture material (adapted here)
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Central dogma of molecular biology
http://www.cem.msu.edu/~reusch/VirtualText/nucacids.htmhttp://www.cem.msu.edu/~reusch/VirtualText/nucacids.htm
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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
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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
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The Genetic Code
Codon usage varies
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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
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Translating sequences
6 possible reading frames, 3 in each direction
AGTCGGCTGACTGCGTTTACGAATGCGATTACTCCCTT
AAGGGAGTAATCGCATTCGTAAACGCAGTCAGCCGACT
Reverse complement
+1
-1
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Translating sequences
6 possible reading frames, 3 in each direction
AGTCGGCTGACTGCGTTTACGAATGCGATTACTCCCTT
AAGGGAGTAATCGCATTCGTAAACGCAGTCAGCCGACT
+2
-2
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Translating sequences
6 possible reading frames, 3 in each direction
AGTCGGCTGACTGCGTTTACGAATGCGATTACTCCCTT
AAGGGAGTAATCGCATTCGTAAACGCAGTCAGCCGACT
-3
+3
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Getting the final protein
Six-frame translation Find longest ORF with initiation site, start
codon and ending with stop codon
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Transcription and translation ATGCGGTGCAACGTGCATCCTAAA
UACGCCACGUUGCACGUAGGAUUU
W G P Y T A K L
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http://www.virtualsciencefair.org/2004/mcgo4s0/public_html/t3/RNA.htmlhttp://www.virtualsciencefair.org/2004/mcgo4s0/public_html/t3/RNA.html
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library.thinkquest.orglibrary.thinkquest.org
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Ribosomes
Protein synthesizers Different subunits for interacting with
mRNA and tRNAs
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Copyright-Anna Kramvis 15
Translation process
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Amino acid structure
The chemistry of R groups distinguishes amino acids and their properties
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Valine Leucine Serine Tyrosine ProlineValine Leucine Serine Tyrosine Proline
Polypeptide chain
Each protein has a unique sequence of amino acids joined into a polypeptide chain
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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.orgcommons.wikimedia.org
www.columbia.eduwww.columbia.edu
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Peptide backbone
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Primary structure, disulphide bonds
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Secondary structure Held together by interactions (H-bonds)
between peptide backbones
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Tertiary structure
Tertiary structure is controlled by the interactions between non-adjacent amino acid R groups
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Quaternary Structure
More than one protein chain, e.g. hemoglobin
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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.
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Other structures
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Summary of protein structures
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The function of a protein depends on sequence of
amino acids and requires a precise folding of its
polypeptide chain
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Properties of Amino Acids
http://www.jalview.org/help/html/misc/properties.gifhttp://www.jalview.org/help/html/misc/properties.gif
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29Copyright-Anna Kramvis
Name R-Group PropertiesGlycine G Gly HydrophobicAlanine A Ala HydrophobicValine V Val HydrophobicLeucine L Leu HydrophobicIsoleucine I Ile Hydrophobic, two chiral carbonsProline P Pro Cyclic, not terribly hydrophobicPhenylalanine F Phe Hydrophobic, bulkyTyrosine Y Tyr Less hydrophobic (than Phe), bulkyTryptophan 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, reactiveThreonine T Thr Hydrophilic, reactive, two chiral carbonsLysine K Lys Highly hydrophilic, positively chargedArginine R Arg Highly hydrophilic, positively chargedHistidine H His Highly hydrophilic, positive or neutralAspartate D Asp Highly hydrophilic, negatively chargedGlutamate E Glu Highly hydrophilic, negatively chargedAsparagine N Asn UnchargedGlutamine Q Gln Uncharged
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Some protein functions
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Information from a protein sequence
MDITIQHPWFKRALGSLYPSRLFDQFFGEGLFEYDLLPFLSSTISPYYRQSLFR
• amino acid composition• molecular weight
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MDQHPWFKRAITIVLLGLLPFLSLYPSRLFDQFCGEGLFEYDSSTISCYRQSLFRTVLESG
Single amino acid physical properties
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
Information from a protein sequence
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MDQHPWFKRAITIVLLGLLPFLSLYCPSRLFDQFCGEGLFEYDSSTISYRQSLFRTNVLES
Functionally important regions
Active site/metal bindingHydrophobic region
disulphide bondGlycosylation site
• Transmembrane regions• Signal sequences• Localisation signals (subcellular location)• Targeting sequences• Modification sites
Information from a protein sequence
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FAMILY
DOMAIN
MOTIF
SITE
RESIDUE
GKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYN
Physical amino acid properties
Conserved domains
Properties of regions
Conserved sequence
Information from a protein sequence
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Protein abundance
Not all genes are expressed all the time, amount of protein is affected by: gene expression -transcriptional regulationPost-transcriptional regulationTranslational regulationPost-translational regulation
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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
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Post-transcriptional regulation
mRNA half-life Antisense RNA RNA splicing siRNAs
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Translational regulation
Ribosomes Translation factors tRNA availability
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Post-translational regulation
Transport to appropriate place Protein folding (chaperones) Post-translational modification:
PhosphorylationAcetylationSugars added….
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http://jp.senescence.info/thoughts/dna_life.jpghttp://jp.senescence.info/thoughts/dna_life.jpg
Summary of main building blocks of biological systems
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Translation exercise
Copyright-Anna Kramvis 41
1. 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?
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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?