Chapter 25 Nucleic Acids and Protein Synthesis

Introduction
Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are the molecules that carry genetic information in the cell

DNA is the molecular archive for protein synthesis RNA molecules transcribe and translate the information from DNA so it can be used to direct protein synthesis

DNA is comprised of two polymer strands held together by hydrogen bonds

Its overall structure is that of a twisted ladder The sides of the ladder are alternating sugar and phosphate units The rungs of the ladder are hydrogen-bonded pairs of heterocyclic amine bases

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 DNA polymers are very long molecules

DNA is supercoiled and bundled into 23 chromosomes for packaging in the cell nucleus

The sequence of heterocyclic amine bases in DNA encodes the genetic information required to synthesize proteins

Only four different bases are used for the code in DNA A section of DNA that encodes for a specific protein is called a gene The set of all genetic information coded by the DNA in an organism is its genome The set of all proteins encoded in the genome of an organism and expressed at any given time is its proteome

The sequence of the human genome is providing valuable information related to human health

Example: A schematic map of genes on chromosome 19 that are related to disease

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 Nucleotides and Nucleosides

Mild degradation of nucleic acids yields monomer units called nucleotides Further hydrolysis of a nucleotide yields:

A heterocyclic amine base D-ribose (from RNA) or 2-deoxy-D-ribose (from DNA); both are C5 monosaccharides A phosphate ion

The heterocylic base is bonded by a b N-glycosidic linkage to C1 of the monosaccharide

Examples: A general structure of an RNA nucleotide (a) and adenylic acid (b)

A nucleoside is a nucleotide without the phosphate group

A nucleoside of DNA contains 2-deoxy-D-ribose and one of the following four bases

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 A nucleoside of RNA contain the sugar D-ribose and one of the four bases adenine, guanine, cytosine or uracil

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 Nucleosides that can be obtained from DNA

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 Nucleosides that can be obtained from RNA

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 Nucleotides can be named in several ways

Adenylic acid is usually called AMP (adenosine monophosphate) It can also be called adenosine 5-monophosphate or 5-adenylic acid

Adenosine triphosphate (ATP) is an important energy storage molecule The molecule 3,5-cyclic adenylic acid (cyclic AMP) is an important regulator of hormone activity

This molecule is biosynthesized from ATP by the enzyme adenylate cyclase

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 Laboratory Synthesis of Nucleosides and

Nucleotides
Silyl-Hilbert-Johnson Nucleosidation

An N-benzoyl protected base reacts with a benzoyl protected sugar in the presence of tin chloride and BSA (a trimethylsilylating agent) The trimethylsilyl protecting groups are removed with aqueous acid in the 2nd step The benzoyl groups can be removed with base

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 Unnatural nucleotide derivatives can be synthesized from nucleosides bearing a substitutable group on the heterocyclic ring

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 Dibenzyl phosphochloridate is a phosphorylating agent for converting nucleosides to nucleotides

The 5-OH is phosphorylated selectively if the 2- and 3-OH groups are protected

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 Deoxyribonucleic Acid: DNA

Primary Structure
The monomer units of nucleic acids are nucleotides

Nucleotides are connected by phosphate ester linkages

The backbone of nucleic acids consists of alternating phosphate and sugar units Heterocyclic bases are bonded to the backbone at each sugar unit The base sequence contains the encoded genetic information The base sequence is always specified from the 5 end of the nucleic acid

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 Secondary Structure
The secondary structure of DNA was proposed by Watson and Crick in 1953 E. Chargaff noted that in DNA the percentage of pyrimidine bases was approximately equal to the percentage of purine bases

Also the mole percentage of adenine Is nearly equal to that of thymine The mole percentage of guanine is nearly equal to cytosine

Chargaff also noted that the ratio of A and T versus G and C varies by species but the ratio is the same for different tissues in the same organism

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 X-ray crystallographic data showed the bond lengths and angles of purine and pyrimidine bases

X-ray data also showed DNA had a long repeat distance (34 )

Based on this data, Watson and Crick proposed the double helix model of DNA (next slide)

Two nucleic acid chains are held together by hydrogen bonding between the bases on opposite strands The double chain is wound into a helix Each turn in the helix is 34 long and involves 10 successive nucleotide pairs Each base pair must involve a purine and a pyrimidine to achieve the proper distance between the sugar-phosphate backbones Base pairing can occur only between thymine and adenine, or cytosine and guanine; no other pairing has the optimum pattern of hydrogen bonding or would allow the distance between sugar-phosphate backbones to be regular

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 Specific pairing of bases means the two chains of DNA are complementary

Knowing the sequence of one chain allows one to also know the sequence of the other

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 Replication of DNA (see next slide)

The DNA strand begins to unwind just prior to cell division Complementary strands are formed along each chain (each chain acts as a template for a new chain) Two new DNA molecules result; one strand goes to each daughter cell

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 RNA and Protein Synthesis

The central dogma of molecular genetics

A gene is the portion of a DNA molecule which codes for one protein

Proteins have many critical functions, e.g., catalysis, structure, motion, cell signaling, the immune response, etc.

DNA resides in the nucleus and protein synthesis occurs in the cytoplasm

Transcription of DNA into messenger RNA (mRNA) occurs in the nucleus mRNA moves to the cytoplasm and the translation into proteins occurs using two other forms of RNA: ribosomal RNA (rRNA) and transfer RNA (tRNA)

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 Transcription: Synthesis of Messenger RNA (mRNA)

In the nucleus a DNA molecule partially unwinds to expose a portion corresponding to at least one gene Ribonucleotides with complementary bases assemble along the DNA strand

Base-pairing is the same in RNA, except that in RNA uracil replaces thymine

Ribonucleotides are joined into a chain of mRNA by the enzyme RNA polymerase

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 An intron (intervening sequence) is a segment of DNA which is transcribed into mRNA but not actually used when a protein is expressed An exon (expressed sequence) in the part of the DNA gene which is expressed Each gene usually contains a number of introns and exons

Introns are excised from mRNA after transcription

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 Ribosomes - rRNA
Protein synthesis is catalyzed in the cytoplasm by ribosomes

A ribosome consists of approximately two thirds RNA and one third protein A ribosome is a ribozyme ( an reaction catalyst made of ribonucleic acid) The 30S subunit binds the mRNA that codes for the protein to be translated The 50S subunit catalyzes formation of the amide bond in protein synthesis

A ribosome has 2 large subunits

Transfer of an amino acid to the growing peptide chain is aided by acid-base catalysis involving an adenine in the 50S subunits

See Figure 25.14, page 1238

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 Transfer RNA (tRNA)

Transfer RNAs (tRNAs), specific to each amino acid, transport amino acids to complimentary binding sites on the mRNA bound to the ribosome

More than one tRNA codes for each amino acid

tRNA is comprised of a relatively small number of nucleotides whose chain is folded into a structure with several loops

One arm of the tRNA always terminates in the sequence cytosine-cytosineadenine, and it is here the amino acid is attached On another arm is a sequence of three bases called the anticodon, which binds with the complementary codon on mRNA

The mRNA genetic code is shown on the next page The structure of a tRNA molecule is shown in Figure 25.15, page 1240

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 The Genetic Code

The genetic code is based on three-base sequences in mRNA Each three-base sequence corresponds to a particular amino acid

The fact that three bases are used to code for each amino acid provides redundancy in the overall code and in the start and stop signals N-formyl methionine (fMet) is the first amino acid incorporated into bacterial protein and appears to be the start signal fMet is removed from the protein chain before its synthesis is complete

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 Translation
Translation is peptide synthesis by a ribosome using the code from an mRNA The following is an example (see figure on next page):

An mRNA binds to a ribosome A tRNA with the anticodon for fMet associates with the fMet codon on the mRNA A tRNA with anticodon UUU brings a lysine residue to the AAA mRNA codon The 50S ribosome catalyzes amide bond formation between the fMET and lysine The ribosome moves down the mRNA chain to the next codon (GUA) A tRNA with the anticodon CAU brings a valine residue The ribosome catalyzes amide bond formation between Lys and Val The ribosome moves along the mRNA chain and the process continues, e.g., with the tRNA for phenylalanine binding to the ribosome A stop signal is reached and the ribosome separates from the mRNA At this point the polypeptide also separates from the ribosome

The polypeptide begins to acquire its secondary and tertiary structure as it is being synthesized Several ribosomes can be translating the same mRNA molecule simultaneously Protein molecules are synthesized only when they are needed

Regulator molecules determine when and if a particular protein will be expressed i.e. synthesized

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 Determining the Base Sequence of DNA

The Chain-Terminating (Dideoxynucleotide) Method

DNA molecules are replicated in such a way that a family of partial copies is generated; each DNA copy differs in length by only one base Random chain-termination is done by poisoning a replication reaction with a low concentration of 23-dideoxynucleotides, which are incapable of chain elongation at their 3 position The 23-dideoxynucleotides are labeled with covalently attached colored fluorescent dye molecules, with each color representing a base type

The partial copies are separated according to length by capillary electrophoresis The terminal base on each strand is detected by the color of laser-induced fluorescence as each DNA molecule passes the detector A four-color chromatogram is generated (see Figure 25.17, page 1246)

Automation of high-throughput dideoxy sequencing made possible completion of the Human Genome Project by the 50th anniversary of Watson and Cricks elucidation of the structure of DNA in 2003
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 Laboratory Synthesis of DNA

Solid-phase methods for laboratory synthesis of DNA are similar to those used for laboratory synthesis of proteins

The solid phase is often controlled-pore glass (CPG) Protecting/blocking reagents are needed (e.g., the dimethoxytrityl and b cyanoethyl groups) A coupling reagent (1,2,3,4-tetrazole) is used to join the protected nucleotides

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 The Polymerase Chain Reaction (PCR)

PCR is an extraordinarily simple and effective method for exponentially multiplying (amplifying) the number of copies of a DNA molecule.

PCR beginning with a single molecule can lead to 100 billion copies in an afternoon The Nobel Prize was awarded to K. Mullis in 1993 for invention of PCR A sample of the DNA to be copied The enzyme DNA polymerase A short primer sequence complimentary to the template DNA A supply of A, C, G, and T nucleotide triphosphate monomers A simple device for thermal cycling during the reaction sequence

PCR requires:

The PCR process is summarized on the next 2 slides

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