CHAPTER 21

NUCLEIC ACIDS AND PROTEIN SYNTHESIS

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What Role Do Nucleic Acids Play?

• • DNA  Contained in cell nucleus   All information needed for the development of a complete living system Every time a cell divides, cell’s DNA is copied and passed to the new cells.

RNA  Part of the process of making proteins from genetic information encoded in DNA  RNA transcribes the information contained in the genes and carries the code out to the protein-making machinery 2

A. Components of Nucleic Acids

• • DNA and RNA are both nucleic acids  Both: unbranched polymers of repeating nucleotide monomers  Each nucleotide has three components: a nitrogenous base, a five-carbon sugar, and a phosphate group.

Nitrogen-containing bases:   Derivatives of pyrimidine or purine.

Adenine (A) and guanine (G) are purines, and cytosine C and thymine (T) are pyrimidines. RNA uses the same bases, except that T is replaced by uracil (U).

Nitrogenous Base Structures 3

The Four DNA Bases, and Uracil

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Ribose/Deoxyribose

• • Both RNA and DNA contain 5-carbon sugars.

 RNA: ribose  DNA: deoxyribose The carbons in the sugars are numbered with primes.

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Nucleosides/Nucleotides

• • Nucleoside: base + sugar Nucleotide: base + sugar + phosphate group

“Tide contains phosphates!”



Naming: Adenine + ribose = adenosine Adenine + deoxyribose = deoxyadenosine Naming nucleosides of the other bases follows the same pattern.

Naming nucleotides: nucleoside name followed by 5’-monophosphate ex. Adenosine 5’-monophosphate (AMP) or deoxyadenosine 5’ monophosphate (dAMP)

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Nucleosides/Nucleotides

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Nucleoside Di- and Triphosphates

• • • Any nucleoside 5’-monophosphate can bind additional phosphate groups, forming a diphosphate or triphosphate For example, you can form the famous ADP (adenosine 5’-diphosphate) and ATP (adenosine 5’-triphosphate) through the addition of phosphate groups.

The same can be done for nucleosides of other bases (ex. GTP, CDP, etc) 8

Nucleoside Di- and Triphosphates

9

Let’s practice…

• Identify each of the following as a nucleoside or a nucleotide:  Guanosine

Nucleoside - “phosphate” not part of the name

 Deoxythymidine 

Nucleoside

Cytidine 5’-monophosphate

Nucleotide - “phosphate” is part of the name

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B. Primary Structure of Nucleic Acids

• • • The nucleotides are linked together from the 3’ -OH of the sugar in one nucleotide to the phosphate on the 5’ carbon of the next nucleotide.

This phosphate link is called a phosphodiester bond. The chain formed from multiple phosphodiester bonds forms the backbone of a strand of DNA.

Phosphodiester bond formation Sequence of bases in the nucleic acid = primary structure. The sequence is written with 5’ and 3’ ends labeled, for instance - 5’-ACGT-3’ 11

A Single Strand of RNA (ACGU)

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C. DNA Double Helix

• In the 1940’s, it was discovered that the percent of A in an organism = % T. Likewise, %C = %G.

What might this suggest?

• • • Base pairing rules: in two complementary strands of DNA, A always base pairs with T, and C always base pairs with G.

1953: DNA discovered to be a double helix (winds like a spiral staircase) DNA Double Helix The strands are antiparallel.

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A DNA Molecule (at least according to the computer)

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D. DNA Replication

• • • • Whenever cells divide, the DNA in the cells needs to replicate -- an exact copy of the DNA needs to be passed to the new cells.

Replication begins when the enzyme

helicase

unwinds a portion of the helix by breaking hydrogen bonds between the strands.

A nucleoside triphosphate bonds to the sugar at the end of the growing new strand. Two phosphate groups are cleaved (this provides the energy for the reaction) And

DNA polymerase

catalyzes the formation of the new phosphodiester bond.

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DNA Replication

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DNA Replication cont.

• • When the entire DNA double helix has been replicated, one strand will be from the original DNA and one will be a newly synthesized strand.

 This is why the process is called semi-conservative replication  Ensures an exact copy of the original DNA through base pairing rules The process of replication has directionality. New nucleotides are only added onto the 3’ end of a growing chain.

  The chain that grows in the 5’ --> 3’ direction: leading strand. Continuously synthesized.

The chain that grows in the 3’ --> 5’ direction: lagging strand.

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How is the lagging strand synthesized?

• • As replication forks (bubbles along the double helix) open up, short fragments of the lagging strand are synthesized in the 5’ --> 3’ direction as space allows. These fragments are called Okasaki fragments.

These fragments are eventually joined by

DNA ligase

to create a continuous strand of DNA.

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Synthesis of Lagging Strand

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E. RNA and Transcription

• 1.

RNA is similar to DNA, except… Different sugar (ribose instead of deoxyribose) 2.

3.

4.

The nitrogen base uracil replaces thymine RNA molecules are single stranded (not double stranded) RNA molecules are much smaller than DNA molecules 20

Three Types of RNA

1.

2.

3.

Ribosomal RNA (rRNA) -- contained in ribosomes, the site of protein synthesis  Messenger RNA (mRNA) Carries genetic info from DNA in nucleus to ribosomes in cytoplasm for protein synthesis  Is a copy of the gene Transfer RNA (tRNA) -- brings the appropriate amino acid to the ribosome during the process of protein synthesis. Each tRNA contains an anticodon (three bases complementing a three-base segment on the mRNA) which allows for match-up with exact amino acid.

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Transcription: Synthesis of mRNA

• • • • Begins with unwinding of a section of the DNA containing the gene needing to be copied Initiation point (signal) for transcription: TATAAA RNA polymerase moves along the template strand in the 3’ to 5’ direction, allowing it to synthesize RNA adding new nucleotides to the 3’ end of the new strand.

When a termination signal is reached, the mRNA is released, and DNA recoils back into its double helix structure.

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Transcription

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Processing of mRNA

• • • • Happens in eukaryotic cells, but not in prokaryotes Eukaryotic genes contain introns -- sections that do not code for protein -- interspersed with coding sections called exons Prokaryotic genes do not contain exons and introns Prior to leaving the nucleus, the eukaryotic mRNA undergoes processing -- introns get snipped out, or spliced.

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mRNA Processing

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Regulation of Transcription

• • • The cell goes not make mRNA randomly. There are certain proteins which are constantly needed, but not very many.

Most mRNA is synthesized in response to cellular needs for a particular protein. Regulation is at the level of transcription.

Prokaryotic cells regulate transcription by means of the operon -- more than one gene under the control of the same regulatory center.

 Control site: promoter (place where RNA polymerase binds) and operator (place where repressor may or may not bind) 26

The lac operon (prokaryotes)

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F. The Genetic Code: Codons

• • • • • A sequence of three bases is called a codon.

Each codon specifies an amino acid in the protein.

All 20 amino acids have their own codon -- some amino acids have more than one.

Three codons specify the stop of protein synthesis - they are UAG, UGA, and UAA.

AUG signals the start of protein synthesis and also encondes the amino acid methionine.

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The Genetic Code

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G. Protein Synthesis: Translation

• • • • Occurs at ribosomes, outside of nucleus tRNA are used to translate each codon into an amino acid Anticodon in the bottom loop is a three-base complement to the codon in the mRNA Amino acid is attached to the stem on the opposite end of the tRNA via an aminoacyl-tRNA synthetase..

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A Single tRNA

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Initiation of Protein Synthesis

• • • • Both ribosomal subunits and an mRNA combine, recognizing the start codon on the mRNA The appropriate tRNA binds to the codon Next, the appropriate tRNA binds to the second codon on the mRNA; a peptide bond is formed between the two neighboring amino acids.

  The first tRNA dissociates The ribosome shifts down the mRNA chain, allowing space for the next tRNA down the line to float in and bind This process continues until a stop codon is reached.

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Termination of Protein Synthesis

• • When the ribosome reaches a stop codon, protein synthesis ends.

The entire complex dissociates, and the peptide is released. The peptide can fold.

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Translation Overview

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H. Genetic Mutations

• • • • • Mutation = change in DNA sequence, altering the amino acid sequence as well Causes of mutation: radiation (X rays/UV light), chemicals called mutagens, perhaps viruses Mutation in somatic cell: body cells resulting from division contain the mutation  Could lead to tumor/cancer Mutation in germ cell (egg or sperm): offspring will contain mutation Mutations can affect function of important enzymes 35

Types of Mutations

• • Replacement of one base with another: substitution mutation  May or may not change the individual amino acid, but no downstream effect Frameshift mutation: base is added to, or deleted from, the sequence. Changes reading frame.

 The amino acid in question is affected, as well as all downstream amino acids (out of frame) 36

Types

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Effect of Mutations

• • If an enzyme, may completely lose activity  Does the mutation change the active site directly?

 If not, does it alter the 3D shape of the protein enough so that the substrate can no longer bind?

A defective protein (due to mutation) may result in genetic disease.

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Practice…

For the following mRNA sequence: 5’-ACA-UCA-CGG-GUA-3’ If a mutation changes UCA to ACA, what happens to the protein?

What happens if the first U is removed from the sequence?

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Genetic Diseases

• • Result of a defective enzyme, resulting from a mutation Example -- albinism  An enzyme normally converts tyrosine to melanin (pigment causing hair/skin color)  If this enzyme is defective, no melanin produced = albinism 40

J. Recombinant DNA

• • • “Cutting and pasting” DNA from the same organism, or from different organisms The resulting DNA is called recombinant Has allowed for the production of human insulin, interferon, human growth hormone… 41

Preparing Recombinant DNA

• 1.

2.

3.

4.

Using

E. coli

(prokaryotic) as an example… some bacteria contain circular DNA called plasmids.

Plasma membranes are dissolved and plasmid DNA isolated A restriction enzyme (recognizes a certain DNA sequence and cuts) cuts through the plasmid Another piece of DNA can be placed into the cut plasmid, and ends sealed The recombinant plasmids can be placed into cells 42

Recombinant DNA Prep

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The Point of Recombinant DNA…

• • If you have a cell containing your recombinant plasmid… when the cell multiplies, each new cell will contain this plasmid If your recombinant plasmid contains a gene (protein) of interest following a promoter, you can stimulate the cells to make large amounts of your protein of interest 44

Polymerase Chain Reaction

• • If you only have one copy (or a few copies) of one gene, this is a method to amplify (make a lot of copies) the gene quickly.

1.

2.

3.

Three steps: Heat your DNA of interest -- the double strands will separate Primers (short sequence complementary to each end) are added -- they anneal to the end of your single strands The addition of DNA polymerase and free nucleotides extends along the single strand, filling in until each double strand is complete.

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PCR

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K. Viruses

• • • Cannot replicate without a host cell Invades the host cell, taking over materials necessary for protein synthesis and growth Viral infection:  Virus inserts its genetic material (DNA or RNA) into host cell   Material is replicated into DNA form The viral DNA is used to make viral proteins via transcription and translation  In some cases, the host cell will lyse, releasing new viral particles 47

Life Cycle of a (Lytic) Virus

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Reverse Transcription

• • • Viruses that use RNA as their genetic material must make viral DNA once inside the host cell It does so via the enzyme

reverse transcriptase

. A virus which contains RNA and uses this process is called a

retrovirus

.

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AIDS/HIV: A Retrovirus

• • • HIV destroys helper T cells (important in the immune response) Thus, AIDS is defined by opportunistic infections Treatments for AIDS?

  Nucleoside analogs: transcription enzymes put false nucleotides into strands, proteins can’t be made Protease inhibitors: HIV protease “chops” the final viral peptide into useable form. If protease blocked, viral proteins are nonfunctional 50