DNA/Protein structure-function analysis and prediction

1. DNA/Protein structure-function analysis and prediction Lecture 12: DNA/RNA structure

2. Central Dogma of Molecular Biology Transcription Translation Replication DNA mRNA 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

3. 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 modifi-cations 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.

4. 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) • Exonswill be spliced together by removal of theIntrons • Poly-adenylation siteimportant for transcription termination (but also: mRNA stability, export mRNA from nucleus etc.)

5. DNA makes mRNA makes Protein

6. 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

7. 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-mutationsBecker-type: milder phenotype, “in frame”- mutations Posture changes during progression of Duchenne muscular dystrophy

8. Nucleic acid basics nucleotide nucleoside • Nucleic acids are polymers • Each monomer consists of 3 moietics

9. Nucleic acid basics (2) • Purines and Pyrimidines can base-pair (Watson- Crick pairs) • A base can be of 5 rings Watson and Crick, 1953

10. Nucleic acid as hetero-polymers • DNA and RNA strands • Nucleosides, nucleotides (Ribose sugar, RNA precursor) (2’-deoxy ribose sugar, DNA precursor) REMEMBER: • DNA = deoxyribonucleotides;RNA = ribonucleotides (OH-groups at the 2’ position) • Note thedirectionality of DNA (5’-3’ & 3’-5’) or RNA (5’-3’) • DNA = A, G, C, T ; RNA = A, G, C, U (2’-deoxy thymidine tri- phosphate, nucleotide)

11. So … RNA DNA

12. 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

13. 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:

14. Genetic diseases: cystic fibrosis • Known since very early on (“Celtic gene”) • Autosomal, recessive, hereditary disease (Chr. 7) • Symptoms: • Exocrine glands (which produce sweat and mucus) • Abnormal secretions • Respiratory problems • Reduced fertility and (male) anatomical anomalies 3,000 20,000 30,000

15. 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.

16. 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 Theoretical Model of NBD1. PDB identifier 1NBD as viewed in Protein Explorer http://proteinexplorer.org Diagram depicting the five domains of the CFTR membrane protein (Sheppard 1999). 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

17. 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)

18. Three dimensional structures of double helices Side view: A-DNA, B-DNA, Z-DNA Space-filling models of A, B and Z- DNA Top view: A-DNA, B-DNA, Z-DNA

19. Major and minor grooves

20. 5’ 3’ 3’ 5’ 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 Same strand stacking cross-strand stacking

21. Types of DNA double helix • Type B major conformation DNA Right-handed helix Long and thin • Type Z minor conformation DNA Left-handed helix Longer and thinner • Type A major conformation RNA minor conformation DNA Right-handed helix Short and broad

22. 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.

23. Non B-DNA Secondary structures • Slipped DNA • Cruciform DNA • Triple helical DNA Hoogsteen basepairs Source: Van Dongen et al. (1999) , Nature Structural Biology6, 854 - 859

24. More Secondary structures • Cloverleaf rRNA structure • RNA pseudoknots 16S rRNA Secondary Structure Based onPhylogenetic Data Source: Cornelis W. A. Pleij in Gesteland, R. F. and Atkins, J. F. (1993) THE RNA WORLD. Cold Spring Harbor Laboratory Press.

25. 3D structures of RNA :transfer-RNA structures • Tertiary structure of tRNA • Secondary structure of tRNA (cloverleaf)

26. Ban et al., Science289 (905-920), 2000 3D structures of RNA :ribosomal-RNA structures • Tertiary structure of large rRNA subunit • Secondary structure of large rRNA (16S)

27. 3D structures of RNA :Catalytic RNA • Tertiary structure of self-splicing RNA • Secondary structure of self-splicing RNA

28. Some structural rules … • Base-pairing is stabilizing • Un-paired sections (loops) destabilize • 3D conformation with interactions makes up for this

29. Final notes • Sense/anti-sense RNAantisense RNA blocks translation through hybridization with coding strand Example. Tomatoes synthesize ethylene in order to ripe. Transgenic tomatoes have been constructed that carry in their genome an artificial gene (DNA) that is transcribed into an antisense RNA complementary to the mRNA for an enzyme involved in ethylene production  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