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CHAPTER 21. NUCLEIC ACIDS AND PROTEIN SYNTHESIS. 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
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CHAPTER 21 NUCLEIC ACIDS AND PROTEIN SYNTHESIS
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
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
Ribose/Deoxyribose • Both RNA and DNA contain 5-carbon sugars. • RNA: ribose • DNA: deoxyribose • The carbons in the sugars are numbered with primes.
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)
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)
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
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’
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.
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.
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.
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.
E. RNA and Transcription • RNA is similar to DNA, except… • Different sugar (ribose instead of deoxyribose) • The nitrogen base uracil replaces thymine • RNA molecules are single stranded (not double stranded) • RNA molecules are much smaller than DNA molecules
Three Types of RNA • 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.
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.
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.
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)
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.
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..
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.
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.
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
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)
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.
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?
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
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…
Preparing Recombinant DNA • 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
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
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. • 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.
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
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.
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
