Everything You MUST Know about Gene Expression (AP Bio Unit 6)

DNA is a double-stranded helix made of nucleotide monomers (deoxyribose sugar, phosphate, nitrogenous base). Strands are anti-parallel with A-T and G-C base pairing. DNA stores information, is replicable, highly stable, and mutable, allowing for evolution. RNA is the hereditary molecule in some viruses and is involved in information transfer (mRNA, tRNA, rRNA) and gene regulation in eukaryotes. Prokaryotic DNA is looped circular chromosomes, while eukaryotic DNA is linear and wrapped around histones. Plasmids are small, extra-chromosomal DNA loops in prokaryotes, often used in genetic engineering for gene transfer and replication, and can confer antibiotic resistance.

DNA replication is semiconservative, meaning each new DNA double helix has one original and one newly synthesized strand. It begins at an origin of replication where helicase unwinds the DNA, forming a replication fork. DNA polymerase synthesizes new DNA strands in the 5' to 3' direction, adding nucleotides to existing strands. Primase lays down RNA primers, which DNA polymerase uses as a starting point. Single-strand binding proteins prevent rewinding. The leading strand is synthesized continuously, while the lagging strand is synthesized discontinuously in Okazaki fragments. DNA polymerase I removes RNA primers and replaces them with DNA, and DNA ligase seals gaps between fragments.

The central dogma of molecular genetics is DNA makes RNA makes protein. A gene is a DNA sequence coding for RNA, then protein. mRNA carries instructions from DNA to ribosomes. rRNA forms catalytic parts of ribosomes, binding amino acids. tRNA brings specific amino acids to ribosomes. Small RNAs regulate eukaryotic gene expression. Transcription creates RNA from a DNA template. RNA polymerase binds to a promoter, reads the DNA template strand (3' to 5'), and synthesizes RNA (5' to 3'). It dissociates at a terminator region. The template strand is non-coding; the complementary coding strand has the same sequence as the mRNA (with U instead of T). In prokaryotes, because there is no nucleus, transcription and translation can occur simultaneously, often with multiple ribosomes forming polysomes on an mRNA strand.

The genetic code translates nucleotide sequences into amino acid sequences. Three RNA nucleotides form a codon, coding for one amino acid. The code is nearly universal, specific, and redundant. mRNA contains codons specifying amino acid order. Ribosomes (composed of rRNA and protein) connect amino acids to form polypeptides. tRNA molecules bring specific amino acids to the ribosome, with an anticodon that matches the mRNA codon and an amino acid binding site. Translation initiation involves the small ribosomal subunit binding to mRNA and moving to the start codon (AUG), where an initiator tRNA (carrying methionine) binds. The large ribosomal subunit then joins. Elongation involves subsequent tRNAs bringing amino acids to the A-site, peptide bond formation between amino acids in the A and P sites, and ribosome translocation along the mRNA. Termination occurs when a stop codon is reached, and a release factor protein binds, causing the ribosome to dissociate and the polypeptide to be released.

Operons are gene clusters transcribed as a single RNA in prokaryotes, allowing for gene regulation. An operon includes structural genes (coding for proteins), an operator (where a repressor binds), a promoter (where RNA polymerase binds), and a regulatory gene (producing the repressor protein). The trp operon, responsible for tryptophan synthesis, is a repressible operon. When tryptophan is absent, the repressor protein cannot bind to the operator, allowing RNA polymerase to transcribe the genes and produce tryptophan. When tryptophan is present, it acts as a co-repressor, binding to the repressor and enabling it to block transcription, thus saving energy. The lac operon, for lactose digestion, is an inducible operon. When lactose is absent, the repressor binds to the operator, preventing transcription. When lactose is present, it binds to the repressor, altering its shape so it cannot bind the operator, allowing transcription of lactose-digesting enzymes. Both are negative feedback systems, where the product of the system leads to its shutdown.

Eukaryotic gene regulation is complex due to multicellularity and large genomes. All cells have the same DNA, but express different genes (differentiation). Gene expression is influenced by the environment. Most eukaryotic DNA is non-coding. Genes can be turned off by being tightly packaged around histones (chromatin structure) and by methylation (addition of methyl groups). Genes are activated by acetylation, which loosens DNA. Epigenetics refers to reversible chemical modifications of DNA or DNA packaging that affect gene expression without altering the DNA sequence; these can be intergenerationally transmitted and drive cell differentiation. Eukaryotic transcription is regulated by regulatory DNA sequences (promoters, enhancers) and proteins (activators, repressors, general transcription factors) that control RNA polymerase binding. Different body tissues can coordinate gene expression through shared regulatory sequences, enabling a single hormone to induce specific changes in various tissues.

Eukaryotic genes contain introns (non-coding sequences) and exons (coding sequences). After transcription, pre-mRNA undergoes processing: a 5' GTP cap and a 3' poly-A tail are added. The 5' cap protects mRNA from degradation, assists in nuclear exit and ribosome binding. The 3' poly-A tail enhances stability and delays degradation. Introns are spliced out, and exons are joined together to form mature mRNA. Alternative splicing allows different combinations of exons to be joined, producing multiple protein variants from a single gene, increasing phenotypic variation. Small RNAs, like microRNAs, play significant regulatory roles in post-transcriptional control. They can lead to RNA silencing by causing degradation or translational arrest of mRNA, thus regulating gene expression.

A mutation is a random change in DNA or a chromosome. A point mutation is a change in a single nucleotide. Silent mutations result in the same amino acid due to the redundancy of the genetic code. Nonsense mutations introduce a stop codon, leading to truncated proteins. Missense mutations change one amino acid to another; their effect depends on the chemical properties of the substituted amino acid. Frameshift mutations (insertions or deletions of nucleotides not in multiples of three) alter the reading frame, leading to extensive missense or premature stop codons. Sickle cell disease is caused by a single missense mutation that changes a nonpolar amino acid to a basic one, leading to hemoglobin aggregation under low oxygen conditions and sickled red blood cells. Mutations can be positive (increase fitness, e.g., loss of pelvic spine in sticklebacks in freshwater), negative (decrease fitness, e.g., sickle cell anemia), or neutral (no effect on phenotype, e.g., in non-coding DNA or silent mutations). Mutations are the raw material for natural selection and evolution. Germline mutations occur in gamete-producing cells and are inherited; somatic mutations occur in other body cells and are not inherited.

Vertical gene transfer (parent to offspring) is distinct from horizontal gene transfer (genes transferred between organisms not related by descent). In bacteria, horizontal gene transfer occurs through: Conjugation (bacterial sex), where plasmids with genes (often for antibiotic resistance) are transferred between cells via a pilus. Transformation, where bacteria pick up DNA fragments or plasmids from their environment and incorporate them into their genome. Transduction, where viruses accidentally transfer bacterial host DNA to other bacteria during their replication cycle. Viral recombination occurs when different viral strains infect the same host, leading to mixing of viral genes and the emergence of new viral strains, which can cause epidemics.

Recombinant DNA is DNA combined from more than one source, often artificially created using restriction enzymes and DNA ligase. Restriction enzymes cut DNA at specific restriction sites, creating sticky ends that can bind to complementary sequences. DNA ligase then forms phosphodiester bonds to join the DNA fragments. This process can create recombinant plasmids (e.g., bacterial plasmid with a human gene). To express human proteins in bacteria, introns must be removed as bacteria cannot splice them out effectively. This can be done by chemically synthesizing DNA based on the protein's amino acid sequence or by using reverse transcriptase to create cDNA from mature mRNA (which already has introns removed). Gel electrophoresis sorts DNA fragments by size and charge, used in DNA fingerprinting and restriction fragment analysis. PCR (polymerase chain reaction) is a cell-free technique to amplify DNA samples by repeatedly heating and cooling DNA with primers, heat-resistant DNA polymerase, and free nucleotides, vastly increasing the amount of DNA. DNA sequencing reveals the specific order of A, T, C, G nucleotides, allowing biologists to determine potential proteins, infer evolutionary relationships, analyze cancer mutations, and monitor viral variants.