Information Storage and Processing in Biological Systems:

A seminar course for the Natural Sciences

Sept. 11 Biological Information, Sept 16 DNA, Gene regulation

Sept 18 Translation and Proteins

Sept 23 Enzymes and Signal transduction

Sept 25 Biochemical Networks

Sept 30 Simple Genetic Networks (Dr. Jacob)

Oct 2

Background

ÿ The Thread of Life. Susan Aldridge. Chapter 2

ÿ Molecular Biology of the Cell. Alberts et al. Garland Press

Suggested further reading

• Protein molecules as computational elements in living cells. D. Bray.Nature. 1995 Jul 27;376(6538):307-12.

• Signaling complexes: biophysical constraints on intracellularcommunication. D. Bray. Annu Rev Biophys Biomol Struct. 1998;27:59-75.

• Metabolic modeling of microbial strains in silico. Ms W. Covert, et al.Trends in Biochemical Sciences Vol.26 ( 2001). 179-186.

• Modelling cellular behaviour. D. Endy & R. Brent. Nature(2001) 409: 391-395.

A - Introduction to Proteins / Translation

• The primary structure is defined as the sequence of amino acids in theprotein. This is determined by and is co-linear to the sequence of bases(triplet codons) in the gene*.

5’---CTCAGCGTTACCAT---3’3’---GAGTCGCAATGGTA---5’

5’---CUCAGCGUUACCAU---3’

N---Leu-Ser-Val-Thr---C

DNA

RNA

PROTEIN

transcription

translation

* - this is not strictly true in most eukaryotic genomes

Structure of Genes In Eukaryotic Organisms

hnRNAheterogeneous nuclear RNA

RNA splicing

Transcription

mRNA

hnRNAheterogeneous nuclear RNA

RNA splicing

Transcription

mRNA

Introns

Structure of Genes In Eukaryotic Organisms

Exons

Structure of Genes In Eukaryotic Organisms

hnRNAheterogeneous nuclear RNA

RNA splicing

Transcription

mRNA

mRNA

AlternativeRNA splicing

Structure of Genes In Eukaryotic Organisms

hnRNAheterogeneous nuclear RNA

RNA splicing

Transcription

mRNA

Control Elements

Structure of Genes In Eukaryotic Organisms

• Coding sequence can be discontinuous and the gene can be composed ofmany introns and exons.

• The control regions (= operators) can be spread over a large region ofDNA and exert action-at-a-distance.

• There can be many different regulators acting on a single gene – i.e. moresignal integration than in bacteria.

• Alternate splicing can give rise to more than one protein product from asingle ‘gene’.

• Predicting genes (introns, exons and proper splicing) is very challenging.

• Because the control elements can be spread over a large segment of DNA,predicting the important sites and their effects on gene expression are notvery feasible at this time.

Translation

Note that many ribosomes can read one message like beads on astring generating many polypeptide chains simultaneously.

• Translation is the synthesis of a polypeptide (protein) chain using the mRNAtemplate.

• Note the mRNA has directionality and is read from the 5’end towards the 3’end.

Translation•The 5’end is defined at the DNA level by the promoter but this does not definethe translation start.

• The translation start sets the ‘register’ or reading frame for the message.

• The end is determined by the presence of a STOP codon (in the correct readingframe).

Schematic Illustration of Translation

Protein Synthesis involves specialized RNA molecules called transfer RNAor tRNA.

The translation start is dependent on:1) a sequence motif called a ribosome binding site (rbs)2) an AUG start codon 5-10 bp downstream from the rbs

Translation Start Position

3’end of 16S rRNA

3’AU //-5’ UCCUCA |||||| 5’-NNNNNNNAGGAGU-N5-10-AUG-//-3’

mRNA rbs start

In bacteria a single mRNA molecule can code for several proteins. Suchmessages are said to be polycistronic. Since the message for all genes insuch a transcript are present at the same concentration (they are on the samemolecule), one might predict that translation levels will be the same for all thegenes. This is not the case: translation efficiency can vary for the differentmessages within a transcript.

Gene 1 Gene 2 Gene 3 Gene 4

Promoter(Start)

Terminator(Stop)

mRNA

DNA

4 genes , 1 message

Polycistronic mRNA

Tar Tap R B Y Z 5000 1000 <100 1000 18000 10000

(Protein monomer per cell)

Translation Efficiency is an important part of gene expression

A single mRNA may encode several proteins. The final level of eachprotein may vary significantly and is a function of:1) translation efficiency2) protein stability

Translation

B – Introduction to Proteins / Characteristics

• The primary structure is defined as the sequence of amino acids in theprotein. This is determined by and is co-linear to the sequence of bases(triplet codons) in the gene*.

5’---CTCAGCGTTACCAT---3’3’---GAGTCGCAATGGTA---5’

5’---CUCAGCGUUACCAU---3’

N---Leu-Ser-Val-Thr---C

DNA

RNA

PROTEIN

transcription

translation

* - this is not strictly true in most eukaryotic genomes

H2NCHCCH3OHO

amino group carboxylic acid

amino acid(alanine)

There are 20 naturally occurring amino acids in proteins, each withdistinctive ‘side chains’ that give them characteristic chemical properties.

H2NCHCCH3OHO

amino group carboxylic acid

amino acid(alanine)

There are 20 naturally occurring amino acids in proteins, each withdistinctive ‘side chains’ that give them characteristic chemical properties.

a-carbon

Amino acids differ in the side chains on the a-carbon.

H2NCHCCH3OHO

amino group carboxylic acid

amino acid(alanine)

There are 20 naturally occurring amino acids in proteins, each withdistinctive ‘side chains’ that give them characteristic chemical properties.

a-carbon

Amino acids differ in the side chains on the a-carbon.

-CH3 (methyl)

H2NCHCCH2OHOHN

H2NCHCCH3OHO

CHCCH2OHOHNH2NCHCCH3HNO

H2O

+

peptide bond

Alanine + Tyrptophan(ala) + (trp)(A) + (W)

Dipeptide(Ala-Trp)

By convention polypeptides arewritten from the N-terminus (amino)to the C-terminus (carboxy)

Alanine ala AArginine arg RAsparagine asn NAspartic acid asp DCysteine cys CGlutamine gln QGlutamic acid glu EGlycine gly GHistidine his HIsoleucine ile ILeucine leu LLysine lys KMethionine met MPhenylalanine phe FProline pro PSerine ser SThreonine thr TTryptophan trp WTyrosine tyr YValine val V

H2NCHCHOHO

HNCOHO

H2NCHCCH2OHOSH

Glycine

Proline

Cysteine

The Newly Synthesized Polypeptide

• The information from DNA‡RNA‡Protein is linear and the finalpolypeptide synthesized will have a sequence of amino acids defined bythe sequence of codons in the message.

• The sequence of amino acids is called the primary structure.

• Secondary structure refers to local regular/repeating structural elements.

• The folded three dimensional structure is referred to as tertiary structure.

Protein function depends on an ordered / defined threedimensional folding. The final three dimensional folded state of the proteinis an intrinsic property of the primary sequence. How the primarysequence defines the final folded conformation is generally referred to asthe Protein Folding Problem.

Primary structure of green fluorescent protein

(single letter AA codes)

SEQUENCE 238AA

26886MW

MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

The primary sequence can be derived directly from the gene sequence butgoing from sequence to structure or sequence to function is not possibleunless there is a related protein for which structure or function is known.Likewise, the structure alone rarely provides information about function(only if the function of a related protein is known).

Projections of the Tertiary Structure of Green Fluorescent Protein

Backbone tracing

Projections of the Tertiary Structure of Green Fluorescent Protein

Backbone tracing

Ile188-Gly189-Asp190-Gly191-Pro192-Val193

Projections of the Tertiary Structure of Green Fluorescent Protein

“Ribbon diagram” showingsecondary structures

Projections of the Tertiary Structure of Green Fluorescent Protein

“Ribbon diagram” showingsecondary structures

Secondary structures

a-helix

Projections of the Tertiary Structure of Green Fluorescent Protein

“Ribbon diagram” showingsecondary structures

Secondary structures

a-helix b-strand

Projections of the Tertiary Structure of Green Fluorescent Protein

“Wireframe” model showingall atoms and chemical bonds.

Ile188-Gly189-Asp190-Gly191-Pro192-Val193

Projections of the Tertiary Structure of Green Fluorescent Protein

“Stick” model showing allatoms and chemical bonds.

“Space filling” model where each atomis represented as a sphere of its Vander Waals radius.

MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELY

Random Coil“Denatured”“Unfolded”

“Native”“Folded”

“folding”

“denaturation”

The final folded three dimensional (tertiary) structure is anintrinsic property of the primary structure.

Primary structure Tertiary Structure

In general, proteins are unstable outside of the celland very sensitive for solvent conditions.

Active site - the region of a protein (enzyme) to which a substrate moleculebinds.• The active site is formed by the three dimensional folding of the peptidebackbone and amino acid side chains. (lock and key / induced fit)• The active site is highly specific in binding interactions (stereochemicalspecificity).

The three dimensional structure of CAP and the cAMP ligand-binding site(Figures 3-45 and 3-55 from Alberts)

Proteins can undergo changes in their three dimensional structure inresponse to changing conditions or interactions with other molecules.This usually alters the ‘activity’ of the protein.

Conformational Change in Protein Structure

Proteins can undergo changes in their three dimensional structure inresponse to changing conditions or interactions with other molecules.This usually alters the ‘activity’ of the protein.

Conformational Change in Protein Structure

Binding of the substrate (glucose) cause the protein (hexokinase)to shift from an open to closed conformation. (Fig. 5-2, Alberts)