Molecular Biology Terms Starting With I
Molecular Biology Glossary: I
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In Vitro Transcription
/ in VEE-troh tran-SKRIP-shun / · Latin: in vitro (in glass) + transcription
In Vitro Transcription is a laboratory technique in which purified RNA polymerase synthesizes RNA from a DNA template outside living cells, producing defined RNA molecules for research and therapeutic use.
A DNA template carrying a bacteriophage promoter, most often the T7, T3, or SP6 sequence, is incubated with the matching phage RNA polymerase and all four ribonucleoside triphosphates to drive RNA synthesis. T7 RNA polymerase is particularly favored because it transcribes at roughly 200 nucleotides per second and can produce thousands of RNA copies from a single template molecule in a few hours. Modified nucleotides such as N1-methylpseudouridine can be incorporated during the reaction to reduce the innate immune response triggered by synthetic RNA, a modification central to the mRNA vaccines developed against SARS-CoV-2.
Each 30-microgram dose of the Pfizer-BioNTech vaccine contains mRNA produced entirely by T7-driven in vitro transcription, scaled to manufacture billions of doses by 2022. Researchers also use the technique to generate radiolabeled or fluorescent RNA probes, ribozymes, and guide RNAs for CRISPR experiments.
In vitro transcription was first demonstrated with purified Escherichia coli RNA polymerase by Jerard Hurwitz and colleagues in 1960, more than a decade before bacteriophage polymerases were isolated. The discovery that a single-subunit phage enzyme could outperform the multi-subunit bacterial enzyme in yield and simplicity transformed the technique into a routine laboratory tool by the 1980s.
Building Blocks of Nucleic Acids →Transcription can happen only inside a cell. Purified RNA polymerase retains full catalytic activity outside the cell and will synthesize RNA from any DNA template carrying the appropriate promoter sequence.
Scientists studying the hepatitis C virus (Hepacivirus C) use in vitro transcription to produce full-length viral RNA genomes from cloned cDNA templates. A single 10-microliter reaction can yield several micrograms of RNA exceeding 9,000 nucleotides, enough to transfect cultured liver cells and initiate a complete viral replication cycle for antiviral drug testing.
Initiation Complex
/ ih-nih-shee-AY-shun KOM-pleks / · Latin: initiare (to begin) + complexus
Initiation Complex is a multi-protein assembly that forms at a promoter or ribosome to begin transcription or translation, positioning the polymerase or ribosome on the template in the correct orientation to start synthesis.
For RNA polymerase II transcription in eukaryotes, assembly begins when TFIID binds the TATA box roughly 25 to 30 base pairs upstream of the transcription start site, followed by sequential recruitment of TFIIA, TFIIB, the polymerase itself, TFIIF, TFIIE, and TFIIH to complete the pre-initiation complex. TFIIH then uses its helicase subunits XPB and XPD to unwind approximately 11 to 15 base pairs of DNA around the start site, creating the transcription bubble needed for the polymerase to begin synthesis. Formation of this assembly is the primary rate-limiting step in gene expression and the main target of transcriptional activators and repressors.
On the translation side, the eukaryotic 43S pre-initiation complex carries the small ribosomal subunit, Met-tRNA, and initiation factors eIF1, eIF1A, eIF2, and eIF3 before it loads onto the 5-prime cap of an mRNA. Structural studies using cryo-electron microscopy have resolved both complexes at near-atomic resolution, revealing how activator proteins contact TFIID subunits to stimulate assembly rates by up to 100-fold.
Bacterial transcription initiation requires only the sigma factor to direct the core RNA polymerase to a promoter, assembling a functional initiation complex from just five protein subunits. The eukaryotic equivalent requires more than 40 polypeptides, a difference that reflects the far greater regulatory demands of a genome compartmentalized within a nucleus.
Translation Biology →Synthesis starts as soon as DNA or mRNA is present. Initiation requires the ordered assembly of specific proteins at defined sequence elements, and the polymerase or ribosome cannot begin synthesis until this assembly is complete.
Building Blocks of Nucleic Acids →During heat-shock responses in the fruit fly Drosophila melanogaster (Drosophila melanogaster), RNA polymerase II pre-initiation complexes assemble at heat-shock gene promoters within seconds of temperature elevation. Genome-wide chromatin immunoprecipitation studies show that these complexes are often pre-loaded in a paused state, poised to release into productive elongation within one to two minutes of the stress signal.
Insertion Sequence
/ in-SER-shun SEE-kwens / · Latin: insertio (a grafting in) + sequentia
Insertion Sequence is a small mobile DNA element found in bacteria that encodes only the transposase enzyme needed to catalyze its own movement between sites in chromosomal or plasmid DNA.
These elements range from 800 to 2,500 base pairs in length and are flanked by short inverted terminal repeats that transposase recognizes to execute the cut-and-paste or replicative transposition reaction. When an insertion sequence lands within or near a promoter, it can reduce gene expression by 5- to 50-fold through transcriptional interference or premature termination. The IS10 element, one of the best-characterized members of the IS4 family, carries 22-base-pair inverted repeats and generates clinically significant antibiotic resistance phenotypes when it inserts near resistance genes in Salmonella enterica and Pseudomonas aeruginosa.
Multiple copies of the same element within 5 to 50 kilobases can align and drive chromosomal deletions or inversions through homologous recombination between the repeated sequences. The laboratory strain Escherichia coli K-12 carries roughly 5 to 8 copies of elements such as IS2, IS3, and IS5, which together account for approximately 0.3 to 0.4 percent of its total chromosome.
The IS1 element, first identified in E. coli in the early 1970s by Heinz Saedler and colleagues, was among the first mobile genetic elements characterized at the molecular level in bacteria. Its discovery helped overturn the assumption that bacterial genomes were static, predating the broader recognition of transposable elements as universal features of genomes across all domains of life.
Bacterial genomes are fixed in order and composition. Insertion sequences move within and between genomes, and their transposition can disrupt genes, alter expression levels, and reorganize chromosome structure on timescales of days to generations.
In Mycobacterium tuberculosis, the insertion sequence IS6110 transposes at variable rates among clinical isolates and is present in 0 to more than 25 copies per genome. Molecular epidemiologists use IS6110 copy number and chromosomal position as a fingerprint to trace tuberculosis transmission chains within outbreaks.