DNA/Protein structure-function analysis and prediction

Lecture 11: DNA/RNA structure

Central Dogma of Molecular Biology

Replication DNATranscription

mRNATranslation

Protein

Transcription is carried out by RNA polymerase (II)

Translation is performed on ribosomes

Replication is carried out by DNA polymerase

Reverse transcriptase copies RNA into DNA

Transcription + Translation = Expression

But DNA can also be transcribed into non-coding RNA …

tRNA (transfer): transfer of amino acids to theribosome during protein synthesis.

rRNA (ribosomal): essential component of the ribosomes (complex with rProteins).

snRNA (small nuclear): mainly involved in RNA-splicing(removal of introns). snRNPs.

snoRNA (small nucleolar): involved in chemical modifications of ribosomal RNAs and other RNA genes. snoRNPs.

SRP RNA (signal recognition particle): form RNA-protein complex involved in mRNA secretion.

Further: microRNA, eRNA, gRNA, tmRNA etc.

RNA editing

Eukaryotes have spliced genes …

Promoter: involved in transcription initiation (TF/RNApol-binding sites) TSS: transcription start site UTRs: un-translated regions (important for translational control) Exons will be spliced together by removal of the Introns Poly-adenylation site important for transcription termination

(but also: mRNA stability, export mRNA from nucleus etc.)

DNA makes mRNA makes Protein

DNA makes mRNA makes Protein

mRNA

Some facts about human genes

There are about 20.000 – 25.000 genes in the human genome (~ 3% of the genome)

Average gene length is ~ 8.000 bp

Average of 5-6 exons per gene

Average exon length is ~ 200 bp

Average intron length is ~ 2000 bp

8% of the genes have a single exon

Some exons can be as small as 1 or 3 bp

DMD: the largest known human gene

The largest known human gene is DMD, the gene that encodes dystrophin: ~ 2.4 milion bp over 79 exons

X-linked recessive disease (affects boys)

Two variants: Duchenne-type (DMD) and Becker-type (BMD)

Duchenne-type: more severe, frameshift-mutations

Becker-type: milder phenotype, “in frame”- mutations

Posture changes during progression of Duchenne muscular dystrophy

Nucleic acid basics

Nucleic acids are polymers

Each monomer consists of 3 moietics

nucleoside

nucleotide

Nucleic acid basics (2)

A base can be of 5 rings Purines and Pyrimidines can base-pair (Watson- Crick pairs)

Watson and Crick, 1953

Nucleic acid as hetero-polymers

Nucleosides, nucleotides

(Ribose sugar,

RNA precursor)

(2’-deoxy ribose sugar,

DNA precursor)

(2’-deoxy thymidine tri-

phosphate, nucleotide)

DNA and RNA strands

REMEMBER:

DNA = deoxyribonucleotides;RNA = ribonucleotides (OH-groups at the 2’ position)

Note the directionality of DNA (5’-3’ & 3’-5’) or RNA (5’-3’)

DNA = A, G, C, T ; RNA = A, G, C, U

So …

DNA RNA

Stability of base-pairing

C-G base pairing is more stable than A-T (A-U) base pairing (why?)

3rd codon position has freedom to evolve (synonymous mutations)

Species can therefore optimise their G-C content (e.g. thermophiles are GC rich) (consequences for codon use?)

Thermocrinis ruber, heat-loving bacteria

DNA compositional biases

Base compositions of genomes: G+C (and therefore also A+T) content varies between different genomes

The GC-content is sometimes used to classify organism in taxonomy

High G+C content bacteria: Actinobacteriae.g. in Streptomyces coelicolor it is 72%

Low G+C content: Plasmodium falciparum (~20%)

Other examples:

Saccharomyces cerevisiae (yeast) 38%

Arabidopsis thaliana (plant) 36%

Escherichia coli (bacteria) 50%

Genetic diseases: cystic fibrosis

Known since very early on (“Celtic gene”)

Autosomal, recessive, hereditary disease (Chr. 7)

Symptoms:

In exocrine glands (which produce sweat and mucus)

Abnormal secretions Respiratory problems Reduced fertility and (male)

anatomical anomalies30,000

3,00020,000

cystic fibrosis (2)

Gene product: CFTR (cystic fibrosis transmembrane conductance regulator)

CFTR is an ABC (ATP-binding cassette) transporter or traffic ATPase.

These proteins transport molecules such as sugars, peptides, inorganic phosphate, chloride, and metal cations across the cellular membrane.

CFTR transports chloride ions (Cl-) ions across the membranes of cells in the lungs, liver, pancreas, digestive tract, reproductive tract, and skin.

cystic fibrosis (3)

CF gene CFTR has 3-bp deletion leading to Del508 (Phe) in 1480 aa protein (epithelial Cl- channel)

Protein degraded in ER instead of inserted into cell membrane

The deltaF508 deletion is the most common cause of cystic fibrosis. The isoleucine (Ile) at amino acid position 507 remains unchanged because both ATC and ATT code for isoleucine

Diagram depicting the five domains of the CFTR membrane protein (Sheppard 1999).

Theoretical Model of NBD1. PDB identifier 1NBD as viewed in Protein Explorer http://proteinexplorer.org

Let’s return to DNA and RNA structure …

Unlike three dimensional structures of proteins, DNA molecules assume simple double helical structures independent on their sequences.

There are three kinds of double helices that have been observed in DNA: type A, type B, and type Z, which differ in their geometries.

RNA on the other hand, can have as diverse structures as proteins, as well as simple double helix of type A.

The ability of being both informational and diverse in structure suggests that RNA was the prebiotic molecule that could function in both replication and catalysis (The RNA World Hypothesis).

In fact, some viruses encode their genetic materials by RNA (retrovirus)

Three dimensional structures of double helices

Side view: A-DNA, B-DNA, Z-DNA

Top view: A-DNA, B-DNA, Z-DNA

Space-filling models of A, B and Z- DNA

Major and minor grooves (1)

Major and minor grooves (2)

The major groove is approximately 50% wider than the minor.

Proteins that interact with DNA often make contact with the edges of the base pairs that protrude into the major groove.

Forces that stabilize nucleic acid double helix

There are two major forces that contribute to stability of helix formation: Hydrogen bonding in base-pairing Hydrophobic interactions in base stacking

5’

5’

3’

3’

Same strand stacking

cross-strand stacking

Types of DNA double helix

Type A

major conformation RNAminor conformation DNA

Right-handed helixShort and broad

Type B

major conformation DNA

Right-handed helixLong and thin

Type Z

minor conformation DNA

Left-handed helixLonger and thinner

Right handed B-DNA

Secondary structures of Nucleic acids

DNA is primarily in duplex form

RNA is normally single stranded which can have a diverse form of secondary structures other than duplex.

Non B-DNA Secondary structures

Cruciform DNA

Triple helical DNA

Slipped DNA

Hoogsteen basepairs

Source: Van Dongen et al. (1999) , Nature Structural Biology  6, 854 - 859

More Secondary structures

RNA pseudoknots Cloverleaf rRNA structure

Source: Cornelis W. A. Pleij in Gesteland, R. F. and Atkins, J. F. (1993) THE RNA WORLD. Cold Spring Harbor Laboratory Press.

16S rRNA Secondary Structure Based onPhylogenetic Data

3D structures of RNA :transfer-RNA structures

Secondary structure of tRNA (cloverleaf)

Tertiary structure of tRNA

3D structures of RNA :ribosomal-RNA structures

Secondary structure of large rRNA (16S)

Tertiary structure of large rRNA subunit

Ban et al., Science 289 (905-920), 2000

3D structures of RNA :Catalytic RNA

Secondary structure of self-splicing RNA

Tertiary structure of self-splicing RNA

Some structural rules …

Base-pairing is stabilizing

Un-paired sections (loops) destabilize

3D conformation with interactions makes up for this

Final notes

Sense/anti-sense RNAantisense RNA blocks translation through hybridization with coding strand

Example. Tomatoes synthesize ethylene in order to ripe. Transgenictomatoes have been constructed that carry in their genome anartificial gene (DNA) that is transcribed into an antisense RNAcomplementary to the mRNA for an enzyme involved in ethyleneproduction tomatoes make only 10% of normal enzyme amount.

Sense/anti-sense peptidesHave been therapeutically usedEspecially in cancer and anti-viral therapy

Sense/anti-sense proteinsDoes it make (anti)sense?Codons for hydrophilic and hydrophobic amino acids on the sense strand may sometimes be complemented, in frame, by codons for hydrophobic and hydrophilic amino acids on the antisense strand. Furthermore, antisense proteins may sometimes interact with high specificity with the corresponding sense proteins… BUT VERY RARE: HIGHLY CONSERVED CODON BIAS