siRNA and miRNA: Generation and Gene Silencing Mechanisms

Messenger RNA (mRNA) is the intermediary that transfers genetic information from the nucleus to the cytoplasm. RNA differs from DNA in several key aspects: it contains ribose sugar (with oxygen) instead of deoxyribose sugar, uses uracil as its nitrogenous base instead of thymine, and is typically single-stranded. RNA nucleotides consist of three components: a nitrogenous base (uracil, adenine, guanine, or cytosine), ribose sugar, and a phosphate group. RNA is composed of a single chain of nucleotides, unlike DNA which has two complementary strands. This single-stranded structure allows RNA to fold into complex three-dimensional shapes necessary for its functions in protein synthesis.

RNA (ribonucleic acid) is a single-stranded molecule essential for protein synthesis. It consists of three components: nitrogenous bases (adenine, guanine, cytosine, and uracil), ribose sugar, and phosphate groups. Unlike DNA, RNA contains uracil instead of thymine. The ribose sugar has hydroxyl groups at both 2' and 3' positions. Nucleotides form through condensation reactions between bases, sugars, and phosphates, creating phosphodiester bonds that link nucleotides into strands. The strand has directionality with a 5' end (free phosphate) and 3' end (free hydroxyl).

RNA (ribonucleic acid) has a single-stranded structure composed of nucleotides. Each nucleotide consists of three components: a five-carbon sugar (ribose), a phosphate group, and a nitrogenous base. The five-carbon sugar is represented by 'S' and the phosphate group by 'P'. The nitrogenous bases are adenine (A), guanine (G), cytosine (C), and uracil (U). RNA differs from DNA in that it contains ribose sugar instead of deoxyribose and uracil instead of thymine. RNA is involved in protein synthesis and gene expression.

RNA is found in different cellular locations: in prokaryotes, it exists in the cytoplasm and ribosomes; in eukaryotes, it is located in the nucleus, mitochondria, chloroplasts, ribosomes, and cytoplasm. Chemically, RNA is a single-stranded molecule composed of nucleotides, each containing a phosphate group, ribose sugar, and a nitrogenous base. The four bases in RNA are adenine, guanine, cytosine, and uracil (replacing thymine found in DNA). Nucleotides connect via phosphodiester bonds, with ester bonds between phosphate and sugar, and glycosidic bonds between sugar and base. Unlike DNA, RNA does not replicate itself but is synthesized from DNA templates.

RNA polynucleotides share the same essential structure as DNA, stabilized by base stacking, hydrogen bonding, and ionic interactions. However, RNA adopts fundamentally different three-dimensional structures because it lacks a complementary strand for base pairing and possesses free 2' hydroxyl groups on its sugar backbone. Unlike DNA's B-form double helix, RNA duplexes resemble A-DNA. RNA forms diverse secondary structures including duplexes (anti-parallel double helices), single-stranded regions, hairpins/stem-loops (self-complementary regions with 180-degree turns), bulges (non-complementary bases within paired regions), internal loops (mismatches on both strands), and junctions (three or four stems meeting). The classic tRNA structure contains three stem-loops, with bases sometimes forming additional intra-loop pairings. The 5S ribosomal RNA exemplifies complex RNA structure, containing G-U, A-A, and U-U non-Watson-Crick base pairs alongside conventional secondary elements.

Gene expression in eukaryotes operates at five levels: DNA, transcriptional, post-transcriptional, translational, and post-translational. Housekeeping genes are constitutively expressed, while regulated genes respond to stimuli. The central dogma describes DNA→RNA→protein flow, with eukaryotes having additional post-transcriptional processing. DNA-level regulation includes epigenetic modifications (acetylation/methylation of histones controlling gene accessibility), gene amplification (increasing gene copies, e.g., dihydrofolate reductase in Methotrexate resistance), and gene rearrangement (recombining gene segments, e.g., immunoglobulin diversity). These mechanisms provide fundamental control over gene expression at the genomic level.

Eukaryotic gene expression is regulated at three distinct levels: transcriptional regulation controls RNA polymerase activity through basal and specific transcription factors to determine how much mRNA is produced; post-transcriptional regulation modifies mRNA after transcription through alternative splicing, nuclear export control, and mRNA stability regulation; post-translational modification processes proteins after translation using proteases to convert inactive zymogens into active proteins. These多层次的调控 mechanisms allow precise control over gene expression and protein function in complex organisms.

Eukaryotic gene regulation occurs at five distinct levels: (1) Transcription level - the primary and most significant level of regulation, (2) Post-transcriptional modification (RNA processing), (3) Transport level (nuclear export), (4) Translation level, and (5) Post-translational modification. Unlike prokaryotic systems where regulation occurs mainly at the transcription level, eukaryotic cells regulate gene expression at multiple stages throughout the entire gene expression pathway.

Eukaryotic gene regulation refers to the control mechanisms that determine whether genes are expressed (transcribed into mRNA and translated into protein) or remain silent. The complete gene expression process involves: transcription (DNA to pre-mRNA), processing (capping, splicing, polyadenylation), transport (nucleus to cytoplasm), translation (mRNA to protein), and post-translational modification. Gene regulation can occur at any of these five levels: transcriptional, processing, transport, translational, and post-translational. The most common and significant level of regulation in eukaryotic cells is at the transcriptional level, where control of RNA synthesis determines whether a gene will be expressed.

Gene expression in eukaryotes is regulated at multiple levels: (1) Transcriptional control (most important); (2) RNA processing control; (3) RNA transport and localization control; (4) Translation control; (5) Protein degradation control. This multi-level regulation allows cells to precisely control when and how much of each protein is produced.

Molecular biology relies on key enzymes: DNA ligase joins DNA fragments by forming phosphodiester bonds; exonucleases digest DNA from ends; DNA polymerase synthesizes DNA in the 5' to 3' direction with proofreading activity; reverse transcriptase synthesizes cDNA from RNA templates; Taq polymerase withstands high temperatures for PCR applications; kinases add phosphate groups; phosphatases remove them. These enzymes enable DNA manipulation, amplification, and analysis.

Enzymes are biological macromolecules that catalyze reactions in living systems. The topic of enzymes is extensive but interconnected, making it easier to understand when studied systematically. This first lesson covers: what enzymes are, a simple example of their function, historical development, and nomenclature including cofactors, coenzymes, and prosthetic groups.

Molecular biology relies on specific enzymes for DNA and RNA manipulation: nucleases (endonucleases, exonucleases, ribonucleases) degrade nucleic acids; DNA polymerase synthesizes new strands; reverse transcriptase converts RNA to DNA; alkaline phosphatase removes phosphate groups; polynucleotide kinase adds phosphate groups; terminal deoxynucleotidyl transferase creates homopolymers; DNA methyltransferase protects DNA from restriction enzymes; and DNA ligase joins fragments by forming phosphodiester bonds.

Living systems are composed of cells, which carry out biochemical activities including metabolism (anabolism and catabolism). Enzymes are biological catalysts that facilitate these reactions by enhancing reaction rates without being consumed. They are highly specific to particular reactions and are large molecules compared to their substrates. Enzymes are almost always proteins, though some RNA molecules called ribozymes also function as enzymes.

This section introduces key enzymes essential for molecular biology techniques. DNA polymerase catalyzes DNA synthesis during replication using DNA as a template. RNA polymerase synthesizes RNA from DNA templates during transcription. Reverse transcriptase (RNA-dependent DNA polymerase) synthesizes DNA from RNA templates, crucial for retroviral replication and cDNA synthesis. DNA ligase forms phosphodiester bonds between DNA fragments, joining broken strands. Nucleases break phosphodiester bonds: endonucleases cleave internal sites, exonucleases remove nucleotides from ends. Alkaline phosphatase removes 5' phosphate groups to prevent self-ligation. DNA methylation adds methyl groups to cytosine bases for gene regulation and protection. Topoisomerases alter DNA topology by introducing transient breaks to relieve supercoiling.