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 the ribosome 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.
rRNA: 4 types of cytoplasmic rRNA, 2 types of mitochondrial rRNA; cytoplasmic rRNAs combine with ribosomal proteins in the nucleus to form pre 40S and pre 60S ribosomal subunits, which will then be exported to the cytoplasm where they will mature and assume their role in protein synthesis. snRNA: snRNAs are always associated with specific proteins, forming small nuclear RiboNucleoProteins (snRNPs). SRP RNA: (SRP) is an RNA-protein complex present in the cytoplasm of cells that binds to the mRNA of proteins that are destined for secretion from the cell. The RNA component of the SRP in eukaryotes is called 4.5S RNA.
microRNA: appears to regulate the expression of messenger RNA (mRNA) molecules.
eRNA: Efference RNA (eRNA) is derived from intron sequences of genes or from non-coding DNA. The function is assumed to be regulation of translational activity gRNA: gRNAs (for guide RNA) are RNA genes that function in RNA editing. Thus far, RNA editing has been found only in the mitochondria of kinetoplastids
tmRNA: tmRNA has a complex structure with tRNA-like and mRNA-like regions. It has currently only been found in bacteria, but is ubiquitous in all bacteria tmRNA recognizes ribosomes that have trouble translating or reading an mRNA and stall, leaving an unfinished protein that may be detrimental to the cell.

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)
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.)

5. DNA makes mRNA makes Protein
The non coding regions, as mentioned earlier, include promoters, transcriptional regulatory sequences, introns and polyadenylation signals.
Post transcriptional processes that modify the initial RNA transcript usually include 5' cap addition, 3' poly A addition, splicing out of introns.
Post translational cleavage of proteins, while rare, can also occur as in the case of insulin and some hormones.
The use of alternative promoters is common and is used to generate cell type specific mRNAs. These alternative promoters may be found within introns of the gene. (

6. Some facts about human genes
There are about – genes in the human genome (~ 3% of the genome)
Average gene length is ~ 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-mutations Becker-type: milder phenotype, “in frame”- mutations
Posture changes during progression
of Duchenne muscular dystrophy

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

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

10. Nucleic acid as hetero-polymers
Nucleosides, nucleotides
DNA and RNA strands
(Ribose sugar,
RNA precursor)
(2’-deoxy ribose sugar,
DNA precursor)
DNA: two complementary and anti-parallel 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
(2’-deoxy thymidine tri-
phosphate, nucleotide)

11. So …
DNA
RNA

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?)
GC: more resistant to denaturation by high temperatures.
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: Actinobacteria e.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%

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
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)
The RNA World referred to an hypothetical stage in the origin of life on Earth. During this stage, proteins were not yet engaged in biochemical reactions and RNA carried out both the information storage task of genetic information and the full range of catalytic roles necessary in a very primitive self-replicating system.

18. Three dimensional structures of double helices
Side view: A-DNA, B-DNA, Z-DNA
The most common DNA structure in solution is the B-DNA. Under conditions of applied force or twists in the DNA, or under low hydration conditions, it can adopt several helical conformations, referred to as the A-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. 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’ 3’
Same strand stacking
cross-strand stacking

21. Types of DNA double helix
Type A
major conformation RNA
minor conformation DNA
Right-handed helix
Short and broad
Type B
major conformation DNA
Right-handed helix
Long and thin
Type Z
minor conformation DNA
Left-handed helix
Longer and thinner
RNA molecules do not have a regular helical structure like DNA. Instead, they can form complicated 3-dimensional structures where the strands can loop back and form intra-strand base-pairs from self-complementary regions along the chain.

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
Cruciform DNA
Slipped DNA
Slipped DNA: During DNA replication (or repair synthesis), one strand transiently dissociates from the other and then reanneals in a misaligned configuration Cause: elongation (if in primer strand), otherwise deletion. Cruciform: reading the upper strand from 5' to 3' is the same as reading the lower strand from 5' to 3'. As a result, each strand of the DNA can self-anneal and the DNA forms a small cruciform structure
Triple helical DNA
Hoogsteen basepairs
Source: Van Dongen et al. (1999) , Nature Structural Biology  6,

24. More Secondary structures
RNA pseudoknots
Cloverleaf rRNA structure
RNA pseudoknots are tertiary structural elements that result when a loop in a secondary structure pairs with a complementary sequence outside the loop
Many regions are self-complementary and capable of forming double helical segments;
Secondary structure is more highly conserved than primary sequence, i.e. complementary mutations evolve to maintain base paring.
16S rRNA Secondary Structure Based on Phylogenetic 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
Secondary structure of tRNA (cloverleaf)
Tertiary structure of tRNA

26. 3D structures of RNA : ribosomal-RNA structures
Secondary structure of large rRNA (16S)
Tertiary structure of large rRNA subunit
Ban et al., Science 289 ( ), 2000
Many regions are self-complementary and capable of forming double helical segments;
Secondary structure is more highly conserved than primary sequence, i.e. complementary mutations evolve to maintain base paring.

27. 3D structures of RNA : Catalytic RNA
Secondary structure of self-splicing RNA
Tertiary structure of self-splicing RNA
Conserved positions of functionally critical residues within the RNA secondary structure revealed the location of the catalytic core
NB.: P=pairing, J=junction

28. Some structural rules …
Base-pairing is stabilizing
Un-paired sections (loops) destabilize
3D conformation with interactions makes up for this
3D conformation with interactions compensate/resolve for these “inconvenients”

29. Final notes
Sense/anti-sense RNA antisense 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 peptides Have been therapeutically used Especially in cancer and anti-viral therapy
Sense/anti-sense proteins Does 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