Structure and forms of dna&rna

Structures and forms of nucleic acids – DNA and RNA

Rashmi Vijay

Composition of DNA

A pentose sugar – deoxy riobose sugar

Nucleotides – A,T,G,C a phosphate

Pentose sugar in DNA

Deoxyribose sugar 4 C atoms and oxygen molecule forms the ring 5th C atom is outside the, part of CH2 group 3 OH groups at positions 1,3,5

Nitrogen Bases

Purines are double ring compounds with 5 membered imidazole ring joined to pyrimidine ring at positions 4’ and 5’ Pyrimidines are single ring compounds with nitrogenous bases at positions 1,3 of a 6 membered benzene ring

Phosphates

Alternate with deoxyribose sugars joined by 3’-C atom of one deoxyribose to 5’-C atom of the other

Nucleosides & Nucleotides

Nucleotide = a nitrogenous (nitrogen-containing) base + a pentose + a phosphate

Nucleoside = a nitrogenous (nitrogen-containing) base + a pentose

Nucleotide Base Pairing

Formation of Phosphodiester bonds to make a polynucleotide strand

The discovery of the structure of DNA by Watson and Crick in 1953 was a momentous event in science, an event that gave rise to entirely new disciplines and influenced the course of many established ones.

Won Nobel Prize

Structure of DNA: Watson & Crick model

Structure of DNA: Watson & Crick model

1. Right handed double helix, wound around central axis with plectonemic coiling.

2. Two polynucloetide strands run antiparallel3. The offset pairing of the two strands creates a major

groove and minor groove on the surface of the duplex4. Phosphate and dRibose forms the back bone of each

polynucloetide strand5. Nitrogen bases are projected inward6. Two polynucloetide strands held together by H- bonds , AT

and GC7. Each base pair tilts 360 and hence has 3600, and each of this

turn has 10 nucleotide base pair8. Bases are place at a distance of 3.4 A0

9. Diameter of the helix 20 A0

10. Molecular weight / unit length = 2x10 6/ micrometr

DNA Is a Double Helix

To shed more light on the structure of DNA, Rosalind Franklin and Maurice Wilkins used the powerful method of x-ray diffraction to analyze DNA fibers.

They showed in the early 1950s that DNA produces a characteristic x-ray diffraction pattern.

In 1953 Watson and Crick postulated a three dimensional model of DNA structure that accounted for all the available data.

Chargaff Rules

Erwin Chargaff developed a chemical technique to measure the amount of each base present in DNA.

Chargaff also observed certain regular relationships among the molar concentrations of the different bases.

These relationships are now called Chargaff’s rules

He found that the base composition of the DNA, defined as the percent G+C, differs among species but is constant in all cells of an organism and within a species.

Chargaff’s rules

DNA Stores Genetic Information The biochemical investigation of DNA began with

Friedrich Miescher, who in the year 1868 isolated a phosphorus-containing substance, which he called “nuclein,” from the nuclei of pus cells (leukocytes) obtained from discarded surgical bandages.

Miescher and many others suspected this substance is in some way with cell inheritance, but the first direct evidence that DNA is the bearer of genetic information came in 1944 through a discovery made by Oswald T. Avery, Colin MacLeod, and Maclyn McCarty.

The Avery-MacLeod-McCarty experiment.

These investigators found that DNA extracted from a virulent (disease-causing) strain of the bacterium Streptococcus pneumoniae, also known as pneumococcus, genetically transformed a nonvirulent strain of this organism into a virulent form.

When injected into mice, the encapsulated strain of pneumococcus is lethal, whereas the nonencapsulated strain, like the heat-killed encapsulated strain, is harmless.

Earlier research by the bacteriologist Frederick Griffith had shown that adding heat-killed virulent bacteria (harmless to mice) to a live nonvirulent strain permanently transformed the latter into lethal, virulent, encapsulated bacteria.

Avery and his colleagues extracted the DNA from heat-killed virulent pneumococci, removing the protein as completely as possible, and added this DNA to non-virulent bacteria.

The DNA gained entrance into the non-virulent bacteria, which were permanently transformed into a virulent strain.

Avery and his colleagues concluded that the DNA extracted from the virulent strain carried the inheritable genetic message for virulence.

Alfred D. Hershey and Martha Chase experiment

A second important experiment provided independent evidence that DNA carries genetic information.

In 1952 Alfred D. Hershey and Martha Chase used radioactive phosphorus (32P) and radioactive sulfur (35S) tracers to show that when the bacterial virus (bacteriophage) T2 infects its host cell, Escherichia coli, it is the phosphorus-containing DNA of the viral particle, not the sulfur-containing protein of the viral coat, that enters the host cell and furnishes the genetic information for viral replication

DNA Can Occur in Different Three-Dimensional Forms

DNA is a remarkably flexible molecule. Many significant deviations from the

Watson-Crick DNA structure are found in cellular DNA, some or all of which may play important roles in DNA metabolism.

These structural variations generally do not affect the key properties of DNA defined by Watson and Crick: strand complementarity, antiparallel strands, and the requirement for A=T and G C base pairs.

Structural variation in DNA reflects three things: the different possible

conformations of the deoxyribose,

rotation about the contiguous bonds that make up the phosphodeoxyribose backbone, and

Free rotation about the C-1–N-glycosyl bond

DNA Can Occur in Different Three-Dimensional Forms

Comparison of A, B, and Z forms of DNA. Each structure shown here has 36 base pairs. The bases are shown in gray, thephosphate atoms in yellow, and the riboses and phosphate oxygens in blue. Blue is the color used to represent DNA strands in later chapters.

Comparison of A, B, and Z forms of DNA.

The Watson-Crick structure is also referred to as B-form DNA, or B-DNA, which is the most stable structure for a random-sequence DNA molecule under physiological conditions and is therefore the standard point of reference in any study of the properties of DNA.

Two structural variants that have been well characterized in crystal structures are the A and Z forms.

Whether A-DNA occurs in cells is uncertain, but there is evidence for some short stretches (tracts) of Z-DNA in both prokaryotes and eukaryotes.

These Z-DNA tracts may play a role (as yet undefined) in regulating the expression of some genes or in genetic recombination.

RiboNucleic Acid (RNA)

Single stranded polymer of nucleotide monomers made of Ribose sugar, Nitrogenous bases and Phosphate group

The structure of RNA is similar to, but not identical with, that of DNA.

There is a difference in the sugar (RNA contains the sugar ribose instead of deoxyribose), RNA is usually single-stranded (not a duplex), and

RNA contains the base uracil (U) instead of thymine (T), which is present in DNA.

Characteristics of RNA

Serves as genetic material in some Viruses

Present in 3 predominant forms rRNA, tRNA and mRNA

Normally doesn't replicate or transcribe

Made of fewer nucleotides (max 12000)

Chain starts with adenine or guanine Contents expressed in terms of

sedimentation coefficients ‘S’ – Svedberg constant

rRNA = forms 80% total cellular RNA tRNA = 10-20% total cellular RNA mRNA = 3-5% total cellular RNA

Ribosomal RNA (rRNA)

Found in Ribosomes most abundant RNA in cells (75%) most stable RNA in cells

Types of rRNA four species rRNA in eukaryotes

28S rRNA (large subunit) 5.8S rRNA (large subunit) 5S rRNA (large subunit) 18S rRNA (small subunit)

28, 18 & 5.8S rRNA are synthesized as a single transcript, then processed -DNA has one promoter, one termination sequence for all three pieces of rRNA -DNA has multiple copies of the whole transcription unit. cut into pieces yielding four spacers & 3 rRNAs. spacers are broken down to nucleotides. rRNA pieces bind ribosomal proteins and begin to self assemble into ribosomal

subunits 5S rRNA transcribed from another gene as a separate transcription unit

three species of rRNA in prokaryotes 23S in large subunit, 5S in the large subunit and 16S in the small subunit

Types of rRNA

Structure of Ribosomal RNA (rRNA)

Made up of nucleotides twisted around itself at some region forming helical structure

The strand assumes a shape of rod, coil or extended strand depending on ionic strength, temp, pH.

In helical region, most of the bases are complementary and are joined by H – bonds

The unpaired/unfolded single strand will have bases, and are not complementary, hence don’t show purine-pyrimidine equality

rRNA strand unfold upon heating and refold on cooling

Secondary structures of rRNA

Secondary structures of rRNA

Messenger RNA (mRNA)

Jacob and Monod coined the term mRNA least abundant (about 5%) of cellular RNAs mRNA may be mono or poly cistronic. bacterial mRNA is not processed eukaryotic mRNA is processed initial eukaryotic transcripts are quite large

allows for posttranscriptional regulation of gene expression

introns, exons and splicing (1) eukaryotic mRNAs contain "introns“(2) introns = intervening sequences (3) exons = expressed sequences(4) introns must be excised from mRNA before translation (5) RNA splicing = process of excising introns from mRNA and

splicing the mRNA back together

mono or poly cistronic mRNA

Structure of mRNA

Single stranded linear molecule without any base pairing (base pairing destroys its nature)

They have complementary sequence to the segment of DNA on which they are transcribed

Regions of mRNA structure

Cap: region at 5’ end. Important for protein synthesis, otherwise mRNA binds poorly to ribosomes. cap protects mRNA from 5'-endonucleases

Non coding region - I (leader): region next to cap with 10-100 nucleotides. Rich in A & U residues and doesn't translate into proteins

Initiation codon: region next NC-I. Common in both Prokaryotes and Eukaryotes

The coding region: consists an average of 1500 nucleotides, which translates in to amino acids

Termination Codon: do not code for any amino acids and thus brings termination of translation

Non coding region – II (trailer): consists about 50-150 nucleotides. doesn't translate into proteins. Contains AAUAA in all sequenced examples

Poly ‘A’ sequence: present at 3’ end. Contains about 200-250 nucleotides, which become shorter with age of the organism. Poly ‘A’ is added in nucleus before m-RNA reaches the cytoplasm from nucleus.

Properties of mRNA

mRNA has rapid turnover. rRNA & tRNA are stable (days,

months) -rRNA deeply buried in structure of ribosomes

both rRNA and tRNA have many modified bases -modified bases help protect against nuclease attack

mRNA turns over fast bacterial mRNA half life = minutes eukaryotic mRNA half life = hours

Properties of mRNA contd. nucleotide composition of mRNA : mRNA

base composition like total genomic DNA base sequence complementarity of mRNA:

measure complementarity by doing RNA-DNA hybridization

size heterogeneity: mRNA varies greatly in size relative to rRNA & tRNA. mRNA varies in size depending on protein for which it codes

gene amplification: one gene (DNA) gives rise to many transcripts (mRNA). each transcript can be translated to many proteins. get more proteins faster

transfer RNA (tRNA)

2nd most abundant (20%) RNA in cells synthesized in precursor form and then processed 16 nucleotide leader sequence removed from 5' end

and terminal UU is removed from 3' end ,UU replaced by CCA

CCA found at the 3' end of all functional tRNAs intron "loop" is removed many bases methylated many bases modified –

uracil converted to dihydrouracil, ribothymine or pseudouridine

adenine converted to inosine

Transfer RNAs vary in length from 73 to 93 nucleotides.

Nucleotide sequence of yeast tRNA Ala. This structure was deduced in 1965 by Robert W. Holley and his colleagues; it is shown in the cloverleaf conformation in which intrastrand base pairing is maximal. The following symbols are used for the modified nucleotides (shaded pink): ,

Ψ, pseudouridine; I, inosine; T, ribothymidine;D, 5,6-dihydrouridine; mI I, 1-methylinosine; m1G, 1-methylguanosine;m2G, N2-dimethylguanosine

Blue lines between parallel sections indicate Watson-Crick base pairs. The anticodon can recognize three codons for alanine (GCA, GCU, and GCC). Note the presence of two G=U base pairs, signified by a blue dot to indicate non-Watson-Crick pairing.In RNAs, guanosine is often basepaired with uridine, although the G=U pair is not as stable as the Watson- Crick G=C pair

Extra nucleotides occur in the extra arm or in the D arm.Two of the arms of a tRNA are critical for its adaptor function. The amino acid arm can carry a specificamino acid esterified by its carboxyl group to the 2- or 3-hydroxyl group of the amino acid residue at the 3 end of the tRNA. The anticodon arm contains the anticodon.The other major arms are the D arm (DHU arm), which contains the unusual nucleotide dihydrouridine (D), and the T Ψ C arm, which contains ribothymidine (T), not usually present in RNAs, and pseudouridine (Ψ), which has an unusual carbon–carbon bond between the base and ribose. The D and T Ψ C arms contribute important interactions for the overall folding of tRNA molecules, and the T Ψ C arm interacts with the large-subunit rRNA.