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Chapter 3 Structures and Functions of Nucleic Acids Nucleic acid A biopolymer composed of nucleotides linked in a linear sequential order through 3’,5’ phosphodiester bonds Classification of nucleic acid • Ribonucleic acid (RNA) is composed of ribonucleotides. – in nuclei and cytoplasm – participate in the gene expression • Deoxyribonucleic acid (DNA) is composed of deoxyribonucleotides. – 90% in nuclei and the rest in mitochondria – store and carry genetic information; determine the genotype of cells Interesting history • 1944: proved DNA is genetic materials (Avery et al.) • 1953: discovered DNA double helix (Watson and Crick) • 1968: decoded the genetic codes (Nirenberg) • 1975: discovered reverse transcriptase (Temin and Baltimore) • 1981: invented DNA sequencing method (Gilbert and Sanger) • 1985: invented PCR technique (Mullis) • 1987: launched the human genome project • 1994: HGP in China • 2001: accomplished the draft map of human genome Section 1 Chemical Components of Nucleic Acids § 1.1 Molecular Constituents Nucleic acid can be hydrolyzed into nucleotides by nucleases. The hydrolyzed nucleic acid has equal quantity of base, pentose and phosphate. phosphate nucleic acid pentose nucleotides nucleosides bases Base: Purine NH2 N N 7 8 9 NH 5 4 6 3 N 1N 2 NH N N Adenine (A) O N NH NH N Guanine (G) NH2 Base: Pyrimidine O 5 4 N 3 2 NH 6 1 NH NH O Uracil (U) NH2 O H3 C N NH Cytosine (C) NH O NH O Thymine (T) Pentose HO CH 2 5´ O OH HO CH 2 OH O 1´ 4´ 3´ OH 2´ OH -D-ribose OH -D-2-deoxyribose Ribonucleoside NH2 N HO CH2 1 O N 1´ O glycosidic bond OH OH Purine N-9 or pyrimidine N-1 is connected to pentose (or deoxypentose) C-1’ through a glycosidic bond. Ribonucleotide NH2 phosphoester bond N O HO P O CH2 O N O OH OH OH A nucleoside (or deoxynucleoside) and a phosphoric acid are linked together through the 5’-phosphoester bond. Nomenclature base nucleoside guanine guanosine cytosine cytidine adenine adenosine uracil uridine nucleotide guanosine monophosphate (GMP) cytidine monophosphate (CMP) adenosine monophosphate (AMP) uridine monophosphate (UMP) (NMP) Nomenclature base nucleoside nucleotide guanine deoxyguanosine deoxyguanosine monophosphate (dGMP) cytosine deoxycytidine deoxycytidine monophosphate (dCMP) adenine deoxyadenosine deoxyadenosine monophosphate (dAMP) thymine deoxythymidine deoxythymidine monophosphate (dTMP) (dNMP) Composition of DNA and RNA Nucleic acid base ribose DNA A、G、C、T deoxyribose RNA A、G、C、U ribose Nucleic acid derivatives Multiple phosphate nucleotides adenosine monophosphate (AMP) adenosine diphosphate (ADP) adenosine triphosphate (ATP) NH NH 22 NH 2 N N N O HO O O PP OO CH OO P CH P HOO PP O CH222 H O O O OH OH OH NN N OO O N N N OH OH OH OH OH OH ADP AMP ATP OH OH OH NN Nucleic acid derivatives Cyclic ribonucleotide: 3’,5’-cAMP, 3’,5’cGMP, used in signal transduction NH2 N O CH2 P O N O cAMP O OH OH N N Nucleic acid derivatives Biologically active systems containing ribonucleotide: NAD+, NADP+, CoA-SH Phosphoester bond formation The -P atom of the triphosphate group of a dNTP attacks the C-3’ OH group of a nucleotide or an existing DNA chain, and forms a 3’phosphoester bond. Nucleic acid chain extension A nucleic acid chain, having a phosphate group at 5’ end and a -OH group at 3’ end, can only be extended from the 3’ end. Phosphodiester bonds Alternative phosphodiester bonds and pentoses constitute the 5’3’ backbone of nucleic acids. Section 2 Structures and Functions of Nucleic Acids § 2.1 Primary Structure • The primary structure of DNA and RNA is defined as the nucleotide sequence in the 5’ – 3’ direction. • Since the difference among nucleotides is the bases, the primary structure of DNA and RNA is actually the base sequence. • The nucleotide chain can be as long as thousands and even more, so that the base sequence variations create phenomenal genetic information. A 5' P C P T P P T C G P P A P P 5' pApCpTpGpCpTpApApC-OH 3' 5' ACTGCTAAC 3' C A P OH 3' § 2.2 Secondary structure The secondary structure is defined as the relative spatial position of all the atoms of nucleotide residues. § 2.2.a Chargaff’s rules • The base composition of DNA generally varies from one species to another. • DNA isolated from different tissues of the same species have the same base composition. • The base composition of DNA in a given species does not change with its age, nutritional state, and environmental variations. • The molarity of A equals to that of T, and the molarity of G is equal to that of C. Molarity of bases A G C T A/T G/C G+C Pu/Py E. coli 26.0 24.9 25.2 23.9 1.09 0.99 50.1 1.04 Tuberc ulosis 15.1 34.9 35.4 14.6 1.03 0.99 70.3 1.00 Yeast 31.7 18.3 17.4 32.6 0.97 1.05 35.7 1.00 Cow 29.0 21.2 21.2 28.7 1.01 1.00 42.4 1.01 Pig 29.8 20.7 20.7 29.1 1.02 1.00 41.4 1.01 Human 30.4 19.9 19.9 30.1 1.01 1.00 39.8 1.01 Historic X-ray diffraction picture Building a milestone of life James Watson and Francis Crick proposed a double helix model of DNA in 1953. It symbolized the new era of modern biology. § 2.2.b Double helix of DNA • Two DNA strands coil together around the same axis to form a right-handed double helix (also called duplex). • The two strands run in opposite directions, i.e., antiparallel. • There are 10 base pairs or 3.4nm per turn and the diameter of the helix is 2.0nm. Antiparallel Backbone and bases The hydrophilic backbone is on the outside of the duplex, and the bases lie in the inner portion of the duplex. Base interactions • The two strands of DNA are stabilized by the base interactions. • The bases on one strand are paired with the complementary bases on another strand through H-bonds, namely G≡C and A=T. • The paired bases are nearly planar and perpendicular to helical axis. • Two adjacent base pairs have base-stacking interactions to further enhance the stability of the duplex. Watson-Crick base pair Watson-Crick base pair Base-stacking interaction Major and minor grooves Groove binding Small molecules like drugs bind in the minor groove, whereas particular protein motifs can interact with the major grooves. § 2.2.c Polymorphisms of DNA • DNA can resume different forms depending upon their chemical microenvironment, such as ionic strength and relative humidity. • B-form DNA is the predominant structure in the aqueous environment of the cells. • A-form and Z-form are also native structures found in biological systems. Structural features of DNAs Feature A-DNA B-DNA Z-DNA Helix rotation Right-handed Right-handed Left-handed Base pair per turn 11 10 12 Pitch 2.46nm 3.4nm 4.56nm Helical diameter 2.55nm 2.0nm 1.84nm Rise per base pair 0.26nm 0.34nm 0.37nm Glycosyl formation Anti- Anti- Anti- at C, syn- at G Rotation between adjacent base pair 33º 36º -60º per dimer Relative humidity 75% 92% Triplet DNA Hoogsteen base pair The third strand is using Hoogsteen Hbonds to pair with bases on the first strand. G-quartet DNA • The telomere of DNA is a G-righ sequence, such as T TG T T G 5’ (TTGGGG)n 3’ • 4 G residues constitute a plane which is stabilized by Hoogsteen H-bonds. T G T T 5' T G 3' G T T G-quartet of DNA Four strands are arranged in either parallel or antiparallel manner. § 2.3 Supercoil Structure § 2.3.a Supercoil structure • The two termini of a linear DNA could be joined to form a circular DNA. • The circular DNA is supercoiled, and supercoil can be either positive or negative. • Only the supercoiled DNA demonstrate biological activities. EM image of supercoiled DNA Circular DNAs in nature, in general, are negatively supercoiled. § 2.3.b Prokaryotic DNA • Most prokaryotic DNAs are supercoiled. • Different regions have different degrees of supercoiled structures. • About 200 nts will have a supercoil on average. § 2.3.c Eukaryotic DNA • DNA in eukaryotic cells is highly packed. • DNA appears in a highly ordered form called chromosomes during metaphase, whereas shows a relatively loose form of chromatin in other phases. • The basic unit of chromatin is nucleosome. • Nucleosomes are composed of DNA and histone proteins. Nucleosome • DNA: ~ 200 bps • Histone: basic proteins with many Lys and Arg residues – H2A (x2), – H2B (x2), – H3 (x2), – H4 (x2) Beads on a string • 146 bp of negatively supercoiled DNA winds 1 ¾ turns around a histone octomer. • H1 histone binds to the DNA spacer. The total length of 46 human chromosomes is about 1.7 m, and becomes 200 nm long after 5 times condensation. § 2.4 Functions of DNA DNA is fundamental to individual life in terms of • They are the material basis of life inheritance, providing the template for RNA synthesis. • They are the information basis for biological actions, carrying the genetic information. • DNA is able to replicate itself in a high fidelity to ensure the genetic information transfer from one generation to the next. • DNA can be used as a template to synthesize RNA (transcription), and RNA is further used as the template to synthesize proteins (translation). • DNA posses the inherent and the mutant properties to create the diversity and the uniformity of the biological world. Gene and genome • A gene is defined as a DNA segment that encodes the genetic information required to produce functional biological products. • A gene includes coding regions as well as non-coding regions. • Genome is a complete set of genes of a given species. Section 3 Structures and Functions of RNA Classification • mRNA (messenger RNA): template for protein synthesis • tRNA (transfer RNA): AA carrier • rRNA (ribosomal RNA): a component of ribosome for protein synthesis • hnRNA (heterogeneous nuclear RNA): precursor of mRNA • snRNA (small nuclei RNA): small RNAs for processing and transporting hnRNA Classes of eukaryotic RNAs Unique features • RNA is single stranded, in general. • RNA has self-complementary intrachain base paring. • The double helical regions of RNA are of the A-form. • RNA is susceptible to hydrolysis. § 3.1 Messenger RNA mRNA is the template for protein synthesis, that is, to translate each genetic codon on mRNA into each AA in proteins. Each genetic codon is a set of three continuous nucleotides on mRNA. • mRNAs constitute 5% of total RNAs. • mRNAs vary significantly in life spans. • hnRNA is the precursor of mRNA. mRNA structure 3'-poly A tail 5'-cap AUG UAA AAA.....AAA coding region 5' non-coding region 3' non-coding region mRNA maturation • hnRNA contains introns and exons. • Exons are the sequences encoding proteins, and introns are non-coding portions. • Splicing process of hnRNA removes introns and makes mRNA become matured. • The matured mRNA has special structure features, including 5’-cap and 3’-poly A tail. 5’-cap OH OH H H H H2N N HN N H 5' O CH2 N O CH3 NH2 N N O O O 5' O P O P O P O CH2 N O N O O O H3' 2'H H H OCH3 O - mRNA chain 5’-cap addition 5’-cap addition • Methylation can occur at different sites on G or A. • 5’-cap can be bound with CBP, benefiting transporting from nucleus to cytoplasm. • 5’-cap can be recognized by translation initiation factor. • It protects the 5’-end from exonucleases. Poly A tail • 20-200 adenine nucleotides at 3’ end • a un-translated sequence. • Related with mRNA degradation that begins with poly A tail shortening. • Associate with poly A tail binding proteins for protection Poly A tailing hnRNA splicing intron exon hnRNA mRNA Matured mRNA of eukaryote 3'-poly A tail 5'-cap AUG UAA AAA.....AAA coding region 5' non-coding region 3' non-coding region § 3.2 Transfer RNA tRNA serves as an amino acid carrier to transport AA for protein synthesis. • tRNA is about 15% of total RNA. • tRNA is 65-100 nucleotides long. • There are at least 20 types of tRNA in one cell. Structure of tRNA • The overall structure is a cloveleaf, reversed L-shape structure. • There are three loops (DHU loop, anticodon loop, TψC loop), and four stems. • The 3-D structure is stabilized by hydrogen bonds of local intrachain base pairs on these stems. Reversed L-shape structure Two key sites of tRNA • A tRNA molecule has an amino acid attachment site and a template-recognition site, bridging DNA and protein. • The template-recognition site is a sequence of three bases called the anticodon complementary to the mRNA codon. Codon and anticodon The anticodon on tRNA pairs with the codon on mRNA. Amino acid attachment • The OH group at the 3' end of tRNA links covalently to an amino acid. • Only the attached AA becomes activated and capable of being transported. Rare Bases tRNA contains a high portion of unusual bases. § 3.3 Ribosomal RNA rRNA provides a proper place for protein synthesis. • rRNA is the most abundant RNA in cells (>80%). • rRNA assembles with numerous ribosomal proteins to form ribosomes. Ribosomes • Ribosomes associate with mRNA to form a place for protein synthesis. • Ribosomes of eukaryotes and prokaryotes are similar in shapes and functions. Components of ribosomes Prokaryote (E.coli) Eukaryote (Liver of mouse) Smaller subunit rRNA proteins 30s 16s 21 40s 1542 nucleotides 18s 40% of total weight 33 1874 nucleotides 50% of total weight Larger subunit rRNA 50s 23s 5s proteins 31 60s 2940 nucleotides 28s 120 nucleotides 5.85s 5s 30% of total weight 49 4718 nucleotides 160nucleotides 120nucleotides 35% of total weight Ribosome of E. coli 23S rRNA 50S large subunit 31 proteins 16S rRNA 70S ribosome 30S small subunit 21 proteins 5S rRNA Secondary structure of 18S rRNA The secondary structure of rRNA has many loops and stems, which can bind ribosomal proteins to form an assembly for protein synthesis. Ribosomal complex 氨基酸 肽链 N末端 退位 进位 E位 A位 核糖体大亚基 核糖体移动方向 m7GpppG AAA...AAA mRNA 核糖体小亚基 P位 Polysomes 核糖体 mRNA 5'末端 3'末端 合成中的多肽 EM of polysomes Section 4 Physical and Chemical Properties of Nucleic Acids General properties • Acidity – Negative backbone • Viscosity – Concentration and aggregation effects • Optical absorption – UV absorption due to aromatic groups • Thermal stability – Disassociation of dsDNA (double-stranded DNA) into two ssDNAs (single-stranded DNA) § 4.1 UV Absorption Application of OD260 Quantify DNAs or RNAs OD260=1.0 equals to 50μg/ml dsDNA 40μg/ml ssDNA (or RNA) 20μg/ml oligonucleotide Determine the purity of nucleic acid samples pure DNA: OD260/OD280 = 1.8 pure RNA: OD260/OD280 = 2.0 Transition of dsDNA to ssDNA The absorbance at 260nm of a DNA solution increases when a dsDNA is melted into two single strands. The change is called hyperchromicity. Melting curve of dsDNA DNA melting • Melting curve: a graphic presentation of the absorbance of dsDNA at 260nm versus the temperature. • Melting temperature (Tm): the temperature at which the UV adsorption reaches the half of the maximum value, also means that about 50% of the dsDNA is disassociated into the single-stranded DNA. Melting curve shift Tm of dsDNA depends on its average G+C content. The higher the G+C content, the higher the Tm. § 4.2 Thermal stability • Dissociation of dsDNA into two ssDNAs is referred to as denaturation. • Denaturation can be partially and completely. • The nature of the denaturation is the breakage of H-bonds. • Denaturation is a common and important process in nature. Denaturation of DNA Extremes in pH or high temperature Cooperative unwinding of DNA strands EM image of denatured DNA Renaturation of DNA Two separated complementary DNA strands can rejoin together to form a double helical form spontaneously when the temperature or pH returns to the biological range. This process is called renaturation or annealing. § 4.3 Hybridization • The ability of DNA to melt and anneal reversibly is extremely important. • An association between two different polynucleotide chains whose base sequences are complementary is referred to as hybridization. • The stability of the hybridized strand depends on the complementary degree. Two dsDNA molecules from different species are completely denutured by heating. When mixed and slowly cooled, complementary DNA strands of each species will associate and anneal to form normal duplexes. • Two ssDNAs, two ssRNAs, as well as one ssDNA and one ssRNA can also be hybridized. • Ionic strength, degree of complementary, temperature, as well as base composition, fragment length of nucleic acids will affect the hybridization. • It is a common phenomenon in biology, and has been used as a convenient techniques in medicine and biology. Target DNA detection • complementary hybridization probe: target: …. TAGCTGAG … …. ATCGACTC … • mismatched hybridization probe: …. TAGCTGAG … non-target: …. ATCAGCTC … Applications • Gene structure and expression • Microarray or gene chip • mRNA separation • Gene diagnosis and therapy • PCR technique Section 5 Nuclease Definition and classification Nucleases are enzymes that are able to hydrolyze phosphoester bonds and cleave DNA or RNA into fragments. • Deoxyribonuclease (DNase) - specially cleave DNA Ribonuclease (RNase) - specially cleave RNA Classification Exonucleases They can cleave terminal nucleotides either from 5’-end or from 3’-end, such as enzymes used in the DNA replication. Endonucleases They can cleave internally at either 3’ or 5’ side of a phosphate group, such as the restriction endonucleases used to construct the recombinant DNA. Exonuclease 5’ 3’ Endonuclease Endonuclease Exonuclease 3’ 5’ Applications • Participate in DNA synthesis and repair, as well as RNA post-translational modification • Digest nucleic acids of food for better absorption • Degrade the invaded nucleic acids • Construct the recombinant DNA