DNA and RNA - University of Sri Jayewardenepura

DNA and RNA - University of Sri Jayewardenepura

DNA and RNA Dr. Sugandhika Suresh Department of Biochemistry Features of the DNA double helix 1. 2 DNA strands per molecule 2. Right-handed helix 3. 2 chains are antiparallel 4. Sugars and phosphates outside 5. Bases inside -- stacked like pennies 6. Bases are bonded together by H-bonds 7. Specific base pairings are observed - complementary 8. A pairs with T

9. C pairs with G 10. 10 base pairs per turn 11. Spacing causes a major and a minor groove Two strands are twisted together around a common axis Right handed Right-Handed vs. Left-Handed Helices

The helix is right-handed As it spirals away from you, the helix turns in a clockwise direction Right- and Left-handed DNA The two strands are antiparallel

One runs in the 5 to 3 direction and the other 3 to 5 Deoxyribose phosphate backbone -hydrophilic Antiparallel DNA Can Form Alternative Types of Double Helices The DNA double helix can form different types of

secondary structure The predominant form found in living cells is B-DNA However, under certain in vitro conditions, A-DNA and Z-DNA double helices can form FORMS OF DNA DNA forms

B-form A-form Z-form A-DNA

Right-handed helix 11 bp per turn Occurs under conditions of low humidity Little evidence to suggest that it is biologically important Z-DNA

Left-handed helix 12 bp per turn Its formation is favored by GG-rich sequences, at high salt concentrations Cytosine methylation, at low salt concentrations Evidence from yeast suggests that it may play a role in transcription and recombination

Denaturation of DNA Denaturation by heating. How is it observed? A260 For dsDNA, The T at which the DNA sample is denatured is called the melting temperature (Tm)

A260=1.0 for 50 g/ml For ssDNA and RNA A260=1.0 for 38 g/ml For ss oligonuleotides A260=1.0 for 33 g/ml Hyperchromic shift If the temperature is lowered, the strands recombine. The rate of reassociation is inversely proportional to the

complexity of the DNA. % Denatured The two strands of the double helix separate reversibly at high temperatures The temperature at which this denaturation or 100 melting occurs depends on the pH and salt 80 concentration, and increases

with the GC content of the 60 DNA. (The curves drawn here are schematic.) 40 40 50 60 70% GC 20 0 70 80 90

100 o Temperature / C 110 Double-stranded and single-stranded DNA differ in their optical absorption at 260 nm dA dG dU dC

The conjugated p-electron systems of the purine & pyrimidine bases absorb strongly in the UV. (Thats why UV light is mutagenic and carcinogenic.) nucleotides ssDNA dsDNA The absorbance of double-stranded DNA (dsDNA) at 260 nm is less than

that of either single-stranded DNA (ssDNA) or the free bases. This is called hypochromism. Importance of Tm Critical importance in any technique that relies on complementary base pairing Designing PCR primers Southern blots Northern blots Colony hybridization Factors Affecting Tm

G-C content of sample Presence of intercalating agents (anything that disrupts H-bonds or base stacking) Salt concentration pH Length Renaturation Strands can be induced to renature (anneal) under proper conditions. Factors to consider: Temperature Salt concentration DNA concentration

Time DNA packaging in chromosomes DNA wound around histone proteins A C T G

C Packaging DNA Histone octamer GA T C A G T G T C A Histone proteins

T A B DNA Helix G C C G T A

2 nm A C T G C Packaging DNA Histone

octamer GA T C A G T G T C A Histone proteins T A B DNA Helix

G C C G T A 2 nm A

C T G C Packaging DNA 11 nm GA T

C A G T G T C A Histone proteins T A B DNA Helix Histone octamer C

C G T Nucleosome G A 2 nm DNA-histone octamer

H1 Links Nucleosomes together Nucleosomes: +H1 -H1 Packaging DNA C A G T G T C A T

A G C C G T A Packaging DNA

C A G T G T C A T A G C C G T A

Packaging DNA Beads on a string A G T C A 11 nm 30 nm

T A G C C G T Tight helical fibre

Looped 200 nm Domains A Protein scaffold Packaging DNA Nucleosomes 11 nm

30 nm T G Tight helical fibre Metaphase Chromosome 700 nm 200 nm Looped Domains C

A 2 nm B DNA Helix Protein scaffold Chromosomes, Chromatids and Centromeres A packaged chromosome Chromatid

Identical chromatid Chromosome arm Centromere Chromosome arm Two identical chromosomes

Replication Anaphase RNA structure and function Objectives The differences between DNA and RNA The structure and function of RNAs RNA & DNA: Similarities Both RNA & DNA: Unbranched polymers Polynucleotides

Contain phosphodiester bonds RNA & DNA: Differences RNA Single-Strand (mostly) Cytoplasm (mainly) AGCU Modified bases Ribose Protein Biosynthesis Post-transcriptional events

DNA Double Nucleus d AGCT Deoxyribose Storage &transfer DNA Repair Biological roles of 1.RNA RNA is the genetic material of some viruses 2. RNA functions as the intermediate (mRNA)

between the gene and the proteinsynthesizing machinery. 3. RNA functions as an adaptor (tRNA) between the codons in the mRNA and amino acids. 4.Through sequence complementarity, RNA serves as a regulatory molecule to bind to and interfere with the translation of certain mRNAs; or as a recognition molecule to guide many posttranscriptional processing steps. 5.Through the tertiary structures, some RNAs function as enzymes to catalyze essential reactions in the cell (RNase P ribozyme, large rRNA in ribosomes, selfsplicing introns, etc). RNA Structure

The primary structure of an RNA strand is much like that of a DNA strand RNA strands are typically several hundred to several thousand nucleotides in length In RNA synthesis, only one of the two strands of

DNA is used as a template Components unique to RNA Replaces Deoxyribose Replaces Thymine 40 RNA Primary Structure

(-e) 5' Structure of RNA backbone (-e) (-e) (-e) 3'

RNA chain directionality: 5'3' Backbone carries charge (-e) on each nucleotide Formation of an RNA structure requires cations Although usually single-stranded, RNA molecules can form short double-stranded regions This secondary structure is due to complementary basepairing

This allows short regions to form a double helix RNA double helices typically A to U and C to G

Are right-handed Have the A form with 11 to 12 base pairs per turn Different types of RNA secondary structures are possible Structures of RNA 1. Primary structure 2.Sequence complementarity: base pairing as DNA 3.Secondary structure

4. Tertiary structure 1. Primary structure RNA RNA contains ribose and uracil and is usually single-stranded STRUCTURE RNA 2.Sequence complementarity: inter- and

intra-molecular base pairing Watson-Crick base pairing STRUCTURE (1) G-C U A-U 3.Secondary structures and interactions

RNA RNA chains fold back on themselves to form local regions of double helix similar2 to A-form structure elements DNA nd STRUCTURE (2)

RNA helix are the basepaired segments between short stretches of complementary sequences, which adopt one of the various stemloop structures hairpin bulge loop

Complementary regions Held together by hydrogen bonds Noncomplementary regions Have bases projecting away from double stranded regions Also called hair-pin The double helical structure of RNA resembles the A-form structure of DNA.

The minor groove is wide and shallow, but offers little sequencespecific information. The major groove is so narrow and deep that it is not very accessible to amino acid side chains from interacting proteins. 4. RNA can fold up into complex tertiary structures Why? RNA has enormous rotational freedom in the backbone of its nonbase-paired regions.

Some RNAs with tertiary structures can catalyze Ribozymes are RNA molecules that adopt complex tertiary structure and serve as biological catalysts. RNase P and self-splicing introns are ribozymes The Central Dogma transcription splicing

mRNA tRNA translation ribosome DNA pre mRNA mRNA

protein RNA Molecules mRNA -messenger tRNA - transfer rRNA - ribosomal Other types of RNA

-RNaseP trimming 5 end of pre tRNA -telomerase RNA- maintaining the chromosome ends -Xist RNA- inactivation of the extra copy of the x chromosome - hn RNA- hetero nuclear - sn RNA small nuclear Messenger RNA (mRNA) codes for protein Small nuclear RNAs (snRNA) splice mRNA in nucleus Transfer RNA (tRNA) carries amino acid to ribosome

Ribosomal RNA (rRNA) is the integral part of the ribosome Small interfering RNA (siRNA) mRNA turn-over, defense mechanism Micro RNA (miRNA) Gene expression regulation RNA: Types Major types: Ribosomal RNA (rRNA) Transfer RNA (tRNA) 80%

15% Messenger RNA (mRNA) 5% The rRNA Nucleoprotein complexes of ribosomes Svedberg Unit: Related to Molecular weight & Shape

The tRNA Smallest RNA 4S (74 95) At least 20 species Unusual bases Secondary structure Intra-chain base pairing Adaptor molecule Carries its sp. a.a. to site of protein biosynthesis tRNA

The mRNA Size:Heterogeneous (500 6000) Primary (precursor): hnRNA Post-transcriptional Processing of Euokaryotic mRNA Carries genetic information from nucleus to cytoplasm (Template of protein synthesis) Reads mRNA

Carries the correct amino acid Essential in translation Has dual specificity since it can read the mRNA and bring the correct AA as well Molecule contains singleand double-stranded regions These spontaneously

interact to produce this 3-D structure tertiary structure of tRNAphe The transfer RNA that carries phenylalanine

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