DNA and RNA Structure

Biotechnology and DNA Technology

• Biotechnology involves manipulating organisms or their components to create useful products.
• Modern biotechnology often refers to DNA technology, which uses lab techniques to study and manipulate genetic material.
• DNA technology allows scientists to extract genes from one organism and transfer them to another.

Recombinant DNA in Genetic Engineering

• Recombinant DNA methods, developed in the 1970s, combine DNA from different sources to create a single molecule in the lab.
• Recombinant DNA technology is used in genetic engineering to manipulate genes for practical applications.
• Genetic engineering includes engineering bacteria to produce chemicals, drugs and pesticides, and transferring genes between bacteria, plants, and animals.

Plasmids and DNA Cloning

• Bacterial plasmids, small circular DNA molecules, replicate independently and are used to manipulate genes in the lab.
• Plasmids carry few genes and can be easily transferred into bacteria.
• Plasmids are inherited by subsequent generations.
• Plasmids are key tools for DNA cloning, which produces many identical copies of a target DNA segment for mass production of useful products.

Gene Cloning Process

• The process involves isolating a specific gene from a longer DNA molecule using gene cloning techniques.
• The first step is to isolate a bacterial plasmid to act as a vector and foreign DNA that contains the gene of interest.
• Foreign DNA can come from various sources, including bacteria, plants, animals, or human tissue cells.
• The plasmid and source DNA are treated with a restriction enzyme that cuts DNA, which cleaves the plasmid at a single site.
• The source DNA is cut into many fragments, with only one fragment carrying the gene of interest.
• Cut DNA from the plasmid and source are mixed, allowing single-stranded ends of the plasmid to base-pair with the complementary ends of the target DNA fragment.
• DNA ligase joins the two DNA molecules, forming covalent bonds between adjacent nucleotides to create a recombinant DNA molecule.
• The recombinant plasmid is mixed with bacteria, which take up the plasmid DNA through transformation.
• The recombinant bacterium reproduces, forming a clone of genetically identical cells carrying the gene of interest.
• A large enough cell clone can be grown to produce the desired protein in marketable quantities.

Purposes of Gene Cloning

• Gene cloning can produce copies of the gene for use in genetic engineering projects
• It can harvest the protein product of the cloned gene for various applications.
• Example: a pest-resistance gene can be cloned from one plant species and transferred into another
• Example: recombinant bacteria can produce medical proteins like insulin in large quantities

Term Definitions

• Biotechnology: The manipulation of living organisms or their components to make useful products.
• Recombinant DNA: A DNA molecule manipulated in the laboratory to carry nucleotide sequences from two sources, often different species.
• Vector: A piece of DNA, usually a plasmid or viral genome, used to move genes from one cell to another.
• Nucleic acid probe: A radioactively or fluorescently labeled single-stranded nucleic acid molecule used to find specific genes or other nucleotide sequences within a mass of DNA.
• Restriction enzymes: Bacterial enzymes that cut up foreign DNA at specific DNA sequences called restriction sites.
• Restriction fragments: Pieces of DNA cut by restriction enzymes.
• Plasmid: A small ring of independently replicating DNA separate from the main chromosome(s) found in prokaryotes and yeasts.
• Gene cloning: The production of multiple copies of a gene.

Enzymes in DNA Manipulation

• Enzymes are used to "cut and paste" DNA in the lab.
• Restriction enzymes are bacterial enzymes that act as cutting tools.
• Each restriction enzyme recognizes a specific short DNA sequence called a restriction site.
• After binding to its restriction site, the enzyme cuts both DNA strands at precise points, creating restriction fragments.
• DNA ligase then joins these fragments together

Restriction Enzymes and Restriction Sites

• EcoRI, a restriction enzyme found in E. coli, recognizes the DNA sequence GAATTC and cuts it at specific sites.
• A restriction site is a short DNA sequence where the restriction enzyme cuts, usually 4-8 nucleotide pairs long.
• Restriction enzymes chop up foreign DNA as a defense mechanism in bacteria
• Bacteria's own DNA is protected by the addition of methyl groups.

Sticky Ends and Complementary Ends

• DNA cut by the same restriction enzyme produces "sticky ends," which are single-stranded extensions from the double-stranded fragments.
• These sticky ends are complementary and can stick together by base pairing.
• Complementary ends on the DNA fragments stick together by base pairing, allowing them to be joined together.

DNA Ligase and Recombinant DNA Formation

• DNA ligase creates new covalent bonds that join the sugar-phosphate backbones of the DNA strands, making the temporary union between the fragments permanent.
• Recombinant DNA is formed by joining restriction fragments from different sources.
• Sticky ends play a key role in this process, where hydrogen bonds form base pairs that hold the strands together.

Genes, Enzymes, and Metabolic Pathways

• In 1902, Archibald Garrod proposed that genes dictate phenotypes through enzymes, which catalyze specific chemical reactions.
• Garrod hypothesized that inherited diseases result from the inability to produce a particular enzyme, citing alkaptonuria as an example.
• Biochemists later supported Garrod's hypothesis, showing that cells use metabolic pathways to synthesize and break down molecules.
• Each step in these pathways is catalyzed by a specific enzyme, and lacking any enzyme prevents completion.
• George Beadle and Edward Tatum demonstrated the relationship between genes and enzymes using Neurospora crassa.
• Each nutritional mutant lacked an enzyme necessary for a metabolic pathway
• They showed that each mutant was defective in a single gene and hypothesized that genes dictate the production of specific enzymes.
• They earned the 1958 Nobel Prize in Chemistry for their work.

Updating the One Gene-One Enzyme Hypothesis

• The "one gene-one enzyme hypothesis" has been updated to include all types of proteins, not just enzymes.
• Proteins such as keratin and insulin are examples of non-enzyme proteins.
• Many proteins are made from two or more polypeptide chains, with each polypeptide specified by its own gene.
• Hemoglobin consists of two kinds of polypeptides, encoded by two different genes.
• Many eukaryotic genes can code for a set of polypeptides through alternative splicing.
• The current definition of a gene is a region of DNA expressed to produce a functional product, either a polypeptide or an RNA molecule.

Cracking the Genetic Code

• Molecular biologists deciphered the genetic code in the 1960s through experimentation.
• An artificial RNA molecule composed solely of uracil (UUU) produced a polypeptide with phenylalanine, confirming UUU specifies phenylalanine.
• Of the 64 codons, 61 code for amino acids.
• AUG codes for methionine and signals the start of a polypeptide chain.
• UAA, UGA, and UAG function as stop codons, marking the end of translation.
• RNA codons have a complementary relationship to DNA codons.
• Codons are arranged linearly in both DNA and RNA, with no gaps.
• There is redundancy in the genetic code, but no ambiguity, as each codon represents only one specific amino acid.

DNA to RNA Translation Example

• DNA segment TAC translates to RNA codon AUG, specifying methionine (Met).
• DNA triplet TTC translates to AAG, designating lysine (Lys).
• The process continues until reaching a stop codon, such as UAG.

Universality of the Genetic Code

• The genetic code is nearly universal across all organisms.
• This enables modern DNA technologies to combine genes from different species.
• It suggests that the genetic code evolved early in the history of life.
• This shared genetic language highlights the evolutionary kinship connecting all life on Earth.

The Process of Transcription in Prokaryotes

• Transcription transfers genetic information from DNA to RNA.
• During transcription, one DNA strand serves as a template for the new RNA molecule, while the other strand is unused.
• RNA polymerase moves along the gene, forming an RNA strand by following base-pairing rules, with U replacing T.
• The process begins at a specific promoter sequence and continues until the enzyme reaches a terminator sequence.

Stages of Gene Transcription

• Initiation: RNA polymerase attaches to the promoter region, opens the double helix, and starts synthesizing RNA.
• Elongation: The RNA strand grows as RNA polymerase moves along the gene; newly formed RNA peels away, and DNA strands come back together.
• Termination: RNA polymerase reaches the terminator DNA sequence and detaches from the RNA and DNA.
• Special DNA sequences mark the start (promoter) and end (terminator) of a gene.

Eukaryotic RNA Processing

• Messenger RNA (mRNA) encodes amino acid sequences and carries genetic messages from DNA to the cell's translation machinery.
• In prokaryotic cells, transcription and translation happen in the cytoplasm.
• In eukaryotic cells, mRNA is transcribed in the nucleus and must travel to the cytoplasm for polypeptide synthesis.
• Eukaryotic transcripts are modified before leaving the nucleus, including the addition of a cap (modified G nucleotide) at the 5' end and a tail (50-250 A nucleotides) at the 3' end.
• These additions facilitate mRNA export, protect it from degradation, and assist ribosomes in binding. The cap and tail themselves are not translated.
• RNA splicing removes noncoding regions called introns and joins coding regions called exons.
• RNA splicing is catalyzed by a complex of proteins and small RNA molecules.
• It allows the production of multiple polypeptides from a single gene by varying the combination of exons included in the final mRNA.
• In humans, RNA splicing enables about 21,000 genes to produce a larger number of polypeptides.
• Eukaryotic genes are longer than mRNA because of introns, which are spliced out of the initial RNA transcript.

Translation Requirements

• Translation converts mRNA into the amino acid language of proteins.
• It requires processed mRNA, enzymes, chemical energy sources like ATP, ribosomes, and transfer RNA (tRNA).

Transfer RNA (tRNA) Function

• Cells use transfer RNA (tRNA) as a molecular interpreter to convert mRNA into proteins.
• tRNA transfers amino acids from the cytoplasm to the growing polypeptide in a ribosome.
• It picks up the correct amino acids and recognizes the corresponding codons in mRNA.
• tRNA is made from a single strand of about 80 nucleotides
• It has a cloverleaf structure with four arms held together by hydrogen bonds.
• The anticodon region varies from one type of tRNA to another.
• tRNAs contain chemically modified bases necessary for proper function.
• The anticodon is a triplet of bases complementary to a codon triplet on mRNA.
• One specific kind of amino acid attaches at the other end of the tRNA molecule.
• There is a slightly different tRNA variety for each amino acid.
• Each amino acid is joined to the correct tRNA by a specific enzyme, with 20 different enzymes for 20 amino acids.
• These enzymes use ATP to bind the appropriate amino acid to its corresponding tRNA.
• The anticodon is the base triplet of a tRNA molecule that couples the tRNA to a complementary codon in the mRNA.

Ribosomes and Protein Synthesis

• Ribosomes coordinate the functioning of mRNA and tRNA and catalyze polypeptide synthesis.
• A ribosome consists of a large subunit and a small subunit, each made up of proteins and ribosomal RNA (rRNA).
• Eukaryotic ribosomes are slightly larger than bacterial ribosomes.
• Antibiotics like tetracycline and streptomycin can target and inactivate bacterial ribosomes.
• The binding site for mRNA is located on the small subunit.
• Binding sites for tRNA (P site and A site) are located on the large subunit.
• Ribosomes hold mRNA and tRNAs together and connect amino acids from the tRNAs to the growing polypeptide chain.

Elongation in Protein Synthesis

• tRNA Binding: The anticodon of an incoming tRNA pairs with the complementary mRNA codon in the ribosome's A site.
• Peptide Bond Formation: The polypeptide detaches from the tRNA in the P site and forms a new peptide bond with the amino acid on the tRNA in the A site.
• Translocation: The tRNA in the P site exits, and the ribosome moves the remaining tRNA from the A site to the P site.
• Elongation continues until a stop codon (UAA, UAG, or UGA) reaches the ribosome's A site.
• The completed polypeptide is released from the last tRNA, and the ribosome disassembles.

Genetic Information Flow

• Genes encode instructions to create RNA molecules, which are then used to produce proteins that control an organism's structures and functions.
• Transcription: mRNA is synthesized from a DNA template.
• In eukaryotic cells, transcription takes place in the nucleus, and the mRNA undergoes processing before moving to the cytoplasm.
• Translation: Occurs in the cytoplasm in four steps.
• The ribosomal subunits separate, and the tRNA and mRNA are released upon polypeptide completion.
• Multiple ribosomes can simultaneously translate the same mRNA molecule.
• Transcription creates a complementary mRNA sequence from a gene's nucleotide sequence in DNA.
• Translation dictates the sequence of amino acids in a polypeptide, which folds into proteins that determine the cell's and organism's appearance and functions.
• A significant portion of the genome is transcribed into other types of RNA that do not code for proteins.

Summary of Protein Synthesis

• Transcription (in the nucleus): RNA polymerase synthesizes mRNA by copying a DNA template.
• Translation (in the cytoplasm):
• Amino acid attachment: Amino acids are linked to specific tRNAs using enzymes and ATP.
• Initiation: The ribosome assembles, and the initiator tRNA binds to the start codon on mRNA.
• Elongation: tRNAs sequentially add amino acids to the growing polypeptide chain as codons are read.
• Termination: The ribosome stops at a stop codon, and the completed polypeptide is released.

Mutations

• Inherited traits can often be explained at the molecular level, such as sickle-cell disease.
• A mutation involves the alteration of one nucleotide pair in the double helix.
• A nucleotide substitution replaces one nucleotide and its base-pairing partner with another pair of nucleotides.
• Silent Mutation: Has no effect on the protein product because both codons code for the same amino acid.
• Missense Mutation: Changes one amino acid to another in a protein; some have little effect, others impair function.
• Nonsense Mutation: Converts an amino acid codon into a stop codon, leading to premature termination.
• Frameshift Mutation: Nucleotides are added or subtracted in a number that is not a multiple of three, altering the reading frame.
• Mutations can arise spontaneously from errors during DNA replication or recombination.
• Mutagens are physical or chemical agents and cause mutations.
• High-energy radiation like X-rays or UV light is a physical mutagen.
• Chemical mutagens disrupt DNA replication.
• AZT mimics thymine, is incorporated into DNA, and blocks further replication
• Mutations create genetic diversity, which is essential for evolution by natural selection.