Chapter 10

Nucleotides and Nucleic Acids Biochemistry by Reginald Garrett and Charles Grisham Garrett and Grisham, Biochemistry, Third Edition

Essential Question

• What are the structures of the nucleotides? How are nucleotides joined together to form nucleic acids? How is information stored in nucleic acids? What are the biological functions of nucleotides and nucleic acids?

Garrett and Grisham, Biochemistry, Third Edition

Outline

• What Is the Structure and Chemistry of Nitrogenous Bases?

• What Are Nucleosides? • What Is the Structure and Chemistry of Nucleotides?

• What Are Nucleic Acids? • What Are the Different Classes of Nucleic Acids?

• Are Nucleic Acids Susceptible to Hydrolysis?

Garrett and Grisham, Biochemistry, Third Edition

Information Transfer in Cells

See Figure 10.1

• Information encoded in a DNA molecule is transcribed via synthesis of an RNA molecule.

• The sequence of the RNA molecule is "read" and is translated into the sequence of amino acids in a protein. Garrett and Grisham, Biochemistry, Third Edition

Figure 10.1

The fundamental process of information transfer in cells.

Information encoded in the nucleotide sequence of DNA is transcribed through synthesis of an RNA molecule whose sequence is dictated by the DNA sequence. As the sequence of this RNA is read (as groups of three consecutive nucleotides) by the protein synthesis machinery, it is

Figure 10.1

sequence of amino acids in a protein. This information transfer system is encapsulated in the dogma: DNA



RNA



protein.

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10.1 – What is the Structure and Chemistry of Nitrogenous Bases?

Know the basic structures

• Pyrimidines – Cytosine (DNA, RNA) – Uracil (RNA) – Thymine (DNA) • Purines – Adenine (DNA, RNA) – Guanine (DNA, RNA) Garrett and Grisham, Biochemistry, Third Edition

Figure 10.2 (a) The pyrimidine ring system; by convention, atoms are numbered as indicated. (b) The purine ring system, atoms numbered as shown. Garrett and Grisham, Biochemistry, Third Edition

Properties of Pyrimidines and Purines

• Keto-enol tautomerism • Acid/base dissociations • Strong absorbance of UV light Garrett and Grisham, Biochemistry, Third Edition

10.2 – What Are Nucleosides?

Know these structures too

• Nucleosides are compounds formed when a base is linked to a sugar. The sugars are pentoses.

• D-ribose (in RNA) • 2-deoxy-D-ribose (in DNA) • The difference - 2'-OH vs 2'-H • This difference affects secondary structure and stability.

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Figure 10.9 Furanose structures —ribose and deoxyribose. Garrett and Grisham, Biochemistry, Third Edition

10.2 – What Are Nucleosides?

• Base is linked via a glycosidic bond.

• The carbon of the glycosidic bond is anomeric.

• Named by adding -idine to the root name of a pyrimidine or -osine to the root name of a purine.

• Conformation can be

syn

or

anti

.

• Sugars make nucleosides more water-soluble than free bases.

• Nucleosides are relatively stable in alkali, but purine nucleosides are easily hydrolyzed in acid to yield the free base and pentose.

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Figure 10.10 b -Glycosidic bonds link nitrogenous bases and sugars to form nucleosides.

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Figure 10.11 The common ribonucleosides —cytidine, uridine, adenosine, and guanosine. Also, inosine drawn in anti conformation. Garrett and Grisham, Biochemistry, Third Edition

Adenosine: a nucleoside with physiological activity

High [Ado] promotes sleepiness. Caffeine blocks the interaction of extracellular Ado with its neuronal receptors.

p. 134 Garrett and Grisham, Biochemistry, Third Edition

10.3 – What Is the Structure and Chemistry of Nucleotides?

Nucleoside phosphates

• Know the nomenclature • "Nucleotide phosphate" is redundant! • Most nucleotides are ribonucleotides • Nucleotides are polyprotic acids.

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Functions of Nucleotides

• Nucleoside 5'-triphosphates are carriers of energy.

• Bases serve as recognition units.

• Cyclic nucleotides are signal molecules and regulators of cellular metabolism and reproduction.

• ATP is central to energy metabolism.

• GTP drives protein synthesis.

• CTP drives lipid synthesis.

• UTP drives carbohydrate metabolism.

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10.4 - What Are Nucleic Acids?

• Polymers linked 3' to 5' by phosphodiester bridges. • Ribonucleic acid and deoxyribonucleic acid. • Know the shorthand notations. • Sequence is always read 5' to 3'.

• In terms of genetic information, this corresponds to "N to C" in proteins.

• The base sequence of a nucleic acid is its distinctive characteristic.

• pGpApCpU, GpApCpUp, pGpApCpUp, GACU, dGACT Garrett and Grisham, Biochemistry, Third Edition

10.5 - What Are the Different Classes of Nucleic Acids?

• DNA - one type, one purpose: - a single DNA molecules in virus and bacteria - Eukaryotic cells have many diploid chromosomes mainly in nucleus, but also mitochondria and chloroplasts.

• RNA - 3 (or 4) types, 3 (or 4) purposes – ribosomal RNA - the basis of structure and function of ribosomes – messenger RNA - carries the message – transfer RNA - carries the amino acids – Small nuclear RNA – Small non-coding RNAs Garrett and Grisham, Biochemistry, Third Edition

The DNA Double Helix

Stabilized by hydrogen bonds!

• DNA: Genetic material: Typically double stranded • dsDNA: Strands antiparallel • Interchain H bonds form base pairs.

• Chargaff’s rules: A=T, G=C, Purines = Pyrimidines • X-ray diffraction of Franklin and Wilkins and model building of Watson and Crick Garrett and Grisham, Biochemistry, Third Edition

The DNA Double Helix

• "Base pairs" arise from hydrogen bonds. • Erwin Chargaff had the pairing data, but didn't understand its implications. • Rosalind Franklin's X-ray fiber diffraction data was crucial. • Francis Crick knew it was a helix. • James Watson figured out the H bonds. Garrett and Grisham, Biochemistry, Third Edition

The sequence of bases in one strand is complementary to the sequence of bases in the other strand.

Figure 10.21 Replication of DNA gives identical progeny molecules because base pairing is the mechanism determining the nucleotide sequence synthesized within each of the new strands during replication.

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The information in DNA is encoded in digital form.

DNA contains two kinds of information: • The base sequences of genes that encode the amino acid sequences of proteins and the nucleotide sequences of functional RNA (rRNA and tRNA).

• The gene regulatory networks that control the expression of protein-encoding (and functional RNA-encoding) genes.

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The Structure of DNA

An antiparallel double helix

• diameter of 2 nm • circular in prokaryotic cells.

• length of 1.6 million nm (

E. coli

) • Compact and folded (

E. coli

cell is only 2000 nm long) • The linear eukaryotic DNA is wrapped around histone proteins to form nucleosomes. • Base pairs: A-T, G-C Garrett and Grisham, Biochemistry, Third Edition

RNA: Typically single stranded: Produced during transcription

• mRNA : Carries information encoded in genes to direct protein synthesis on ribosomes – Derive from heterogeneous nuclear RNA (hnRNA) – RNA processed by splicing (removal of introns and joining of exons), capping (5’ end), and polyA tail addition (3’ end) • rRNA : Components of ribosome: Protein synthesis – Small subunit of ribosome: Single rRNA – Large subunit of ribosome: Large subunit rRNA, 5S rRNA, and in eukaryotes 5.8S rRNA • tRNA : Carriers of activated amino acids used by ribosome for protein synthesis • snRNA: Small nuclear RNAs • siRNAs: Small interfering RNAs: Degrade mRNAs • miRNAs: Bind to mRNA and block translation • snoRNAs: Required for certain RNA modification Garrett and Grisham, Biochemistry, Third Edition

Messenger RNA

Transcription product of DNA

• In prokaryotes, a single mRNA contains the information for synthesis of many proteins.

• In eukaryotes, a single mRNA codes for just one protein, but structure is composed of introns and exons.

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Eukaryotic mRNA

• DNA is transcribed to produce heterogeneous nuclear RNA (hrRNA).

– mixed introns and exons with poly A – intron - intervening sequence – exon - coding sequence – poly A tail - stability? • Splicing produces final mRNA without introns.

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Ribosomal RNA

• Ribosomes are about 2/3 RNA, 1/3 protein.

• rRNA serves as a scaffold for ribosomal proteins. rRNA has a characteristic secondary structure due to many intramolecular H-bonds.

• 23S rRNA in

E. coli

is the peptidyl transferase!

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Ribosomal RNA

• rRNA: Components of ribosome: Protein synthesis –Small subunit of ribosome: Single rRNA –Large subunit of ribosome: Large subunit rRNA, 5S rRNA, and in eukaryotes 5.8S rRNA Garrett and Grisham, Biochemistry, Third Edition

Transfer RNA

• Small polynucleotide chains - 73 to 94 residues each • Several bases are usually methylated.

• Carriers of activated amino acids used by ribosome for protein synthesis.

• Each a.a. has at least one unique tRNA which carries the a.a. to the ribosome.

• 3'-terminal sequence is always CCA-a.a. • Aminoacyl tRNA molecules are the substrates of protein synthesis.

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Structure of a tRNA molecule Garrett and Grisham, Biochemistry, Third Edition

Small RNAs

Altering gene expression in response to stressful environmental situations

• snRNA: Small nuclear RNAs: Important in converting hnRNA to mature mRNA.

• siRNAs: Small interfering RNAs: Degrade mRNAs — post-transcriptional gene silencing • miRNAs: micro RNAs: Bind to mRNA and block translation • snoRNAs: small nucleolar RNAs: Required for certain RNA modifications Garrett and Grisham, Biochemistry, Third Edition

DNA & RNA Differences?

Why does DNA contain thymine?

• Cytosine spontaneously deaminates to form uracil.

• Repair enzymes recognize these "mutations" and replace these uracils with cytosines.

• But how would the repair enzymes distinguish natural U from mutant U.

• Nature solves this dilemma by using thymine (5-methyl-U) in place of uracil.

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DNA & RNA Differences?

Why is DNA 2'-deoxy and RNA is not?

• Vicinal -OH groups (2' and 3') in RNA make it more susceptible to hydrolysis.

• DNA, lacking 2'-OH is more stable.

• This makes sense - the genetic material must be more stable.

• RNA is designed to be used and then broken down.

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Hydrolysis of Nucleic Acids

• RNA is resistant to dilute acid.

• DNA is depurinated by dilute acid.

• DNA is not susceptible to base.

• RNA is hydrolyzed by dilute base.

• See Figure 10.29 for mechanism.

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Peptide Nucleic Acids (PNAs) are synthetic mimics of DNA and RNA

• Poor substrates for proteases • Neutral charge; facilitate penetration into negatively charged cell membranes.

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p.331

Restriction Enzymes

• Bacteria have learned to "restrict" the possibility of attack from foreign DNA by means of "restriction enzymes“.

• Type II and III restriction enzymes cleave DNA chains at selected sites.

• Enzymes may recognize 4, 6 or more bases in selecting sites for cleavage.

• An enzyme that recognizes a 6-base sequence is a "six cutter“.

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Type II Restriction Enzymes

• No ATP requirement.

• Recognition sites in dsDNA have a 2-fold axis of symmetry.

• Cleavage can leave staggered or "sticky" ends or can produce "blunt” ends.

• Names use 3-letter italicized code: • • 1st letter - genus; 2nd,3rd - species • Following letter denotes strain

Eco

RI is the first restriction enzyme found in the R strain of

E. coli.

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