Transcript Lect4 Proteins

PROTEINS
Nicky Mulder
Acknowledgements: Anna Kramvis for lecture
material (adapted here)
Central dogma of molecular biology
http://www.cem.msu.edu/~reusch/VirtualText/nucacids.htm
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

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

The Genetic Code
Codon
usage
varies
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

Translating sequences

6 possible reading frames, 3 in each
direction
AGTCGGCTGACTGCGTTTACGAATGCGATTACTCCCTT
+1
Reverse complement
AAGGGAGTAATCGCATTCGTAAACGCAGTCAGCCGACT
-1
Translating sequences

6 possible reading frames, 3 in each
direction
AGTCGGCTGACTGCGTTTACGAATGCGATTACTCCCTT
+2
AAGGGAGTAATCGCATTCGTAAACGCAGTCAGCCGACT
-2
Translating sequences

6 possible reading frames, 3 in each
direction
AGTCGGCTGACTGCGTTTACGAATGCGATTACTCCCTT
+3
AAGGGAGTAATCGCATTCGTAAACGCAGTCAGCCGACT
-3
Getting the final protein
Six-frame translation
 Find longest ORF with initiation site, start
codon and ending with stop codon

Transcription and translation

ATGCGGTGCAACGTGCATCCTAAA

UACGCCACGUUGCACGUAGGAUUU

W
G
P
Y
T
A
K
L
http://www.virtualsciencefair.org/2004/mcgo4s0/public_html/t3/RNA.html
library.thinkquest.org
Ribosomes
Protein synthesizers
 Different subunits for interacting with
mRNA and tRNAs

Translation
process
Copyright-Anna Kramvis
15
Amino acid structure

The chemistry of
R groups
distinguishes
amino acids and
their properties
Polypeptide chain

Each protein has a unique sequence of
amino acids joined into a polypeptide
chain
Valine
Leucine
Serine
Tyrosine
Proline
Protein primary structure

Proteins made up amino acids joined by
peptide bonds between carboxyl group of
one and amino group of the next
www.columbia.edu
commons.wikimedia.org
Peptide
backbone
Primary structure, disulphide bonds
Secondary structure

Held together by interactions (H-bonds)
between peptide backbones
Tertiary structure

Tertiary structure
is controlled by
the interactions
between nonadjacent amino
acid R groups
Quaternary Structure
More than one
protein chain,
e.g. hemoglobin
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.
Other
structures
Summary
of protein
structures
The function of a protein
depends on sequence of
amino acids and requires a
precise folding of its
polypeptide chain
Properties of Amino Acids
http://www.jalview.org/help/html/misc/properties.gif
Name
Glycine
Alanine
Valine
Leucine
Isoleucine
Proline
Phenylalanine
Tyrosine
Tryptophan
Cysteine
Methionine
Serine
Threonine
Lysine
Arginine
Histidine
Aspartate
Glutamate
Asparagine
Glutamine
G
A
V
L
I
P
F
Y
W
C
M
S
T
K
R
H
D
E
N
Q
Gly
Ala
Val
Leu
Ile
Pro
Phe
Tyr
Trp
Cys
Met
Ser
Thr
Lys
Arg
His
Asp
Glu
Asn
Gln
R-Group Properties
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic, two chiral carbons
Cyclic, not terribly hydrophobic
Hydrophobic, bulky
Less hydrophobic (than Phe), bulky
Hydrophobic, bulky (indole ring)
Hydrophobic, highly reactive (S-S link)
Hydrophobic (start a.a.)
Hydrophilic, reactive
Hydrophilic, reactive, two chiral carbons
Highly hydrophilic, positively charged
Highly hydrophilic, positively charged
Highly hydrophilic, positive or neutral
Highly hydrophilic, negatively charged
Highly hydrophilic, negatively charged
Uncharged
Uncharged
Copyright-Anna Kramvis
29
Some protein functions
Information from a protein sequence
MDITIQHPWFKRALGSLYPSRLFDQFFGEGLFEYDLLPFLSSTISPYYRQSLFR
• amino acid composition
• molecular weight
Information from a protein sequence
Single amino acid physical properties
MDQHPWFKRAITIVLLGLLPFLSLYPSRLFDQFCGEGLFEYDSSTISCYRQSLF
RTVLESG
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
Functionally important regions
MDQHPWFKRAITIVLLGLLPFLSLYCPSRLFDQFCGEGLFEYDSSTISYRQSLF
RTNVLES
Active site/metal
Hydrophobic region
binding
Glycosylation site
disulphide bond
• Transmembrane regions
• Signal sequences
• Localisation signals (subcellular location)
• Targeting sequences
• Modification sites
Information from a protein sequence
FAMILY
DOMAIN
Conserved sequence
Conserved domains
MOTIF
Properties of regions
SITE
RESIDUE
Physical amino acid
properties
GKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYN
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
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

Post-transcriptional regulation
mRNA half-life
 Antisense RNA
 RNA splicing
 siRNAs

Translational regulation
Ribosomes
 Translation factors
 tRNA availability

Post-translational regulation
Transport to appropriate place
 Protein folding (chaperones)
 Post-translational modification:

 Phosphorylation
 Acetylation
 Sugars
added….
Summary of main building
blocks of biological systems
http://jp.senescence.info/thoughts/dna_life.jpg
Translation exercise
1. Translate this mRNA using the genetic code table
5’AUGUUUUUGUCGUACUGGUGUCUACCUCAUCAACGU
AUUACGAAUAAG3’
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
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?


