Gene Expression, RNA Interference, and Protein Translation

Vocabulary flashcards covering RNA detection methods, RNA interference, the genetic code, translation initiation, protein phosphorylation, ubiquitination, autophagy, bioluminescent reporters, and induced pluripotent stem cells.

Threshold (qRT-PCR)

The designated amount of fluorescence detected above background levels, utilized to determine the cycle threshold ($C_t$ value) for exponential gene amplification.

Northern Blot

A molecular biology technique used to detect specific RNA molecules by extracting total RNA, separating the fragments via gel electrophoresis, and transferring them onto a stable nylon or nitrocellulose membrane.

DNA Microarray

A high-throughput solid-surface assay allowing the simultaneous quantitative analysis of thousands of genes by hybridizing labeled target RNAs to complementary pre-fixed probe sequences.

Microarray Expression Signals

Fluorescent signal readouts indicating differential gene regulation, where a green signal denotes upregulation and a red signal denotes downregulation of target genes.

Biotinylated Probes

Single-stranded nucleic acid sequences tagged with biotin molecules to facilitate probe purification and hybridization detection with target RNAs.

RNA Interference (RNAi)

A conserved biological regulatory process in which double-stranded RNA (dsRNA) molecules trigger sequence-specific gene silencing and suppression of gene expression.

Co-suppression

The phenomenon observed in Petunia plants where introducing a chimeric chalcone synthase transgene led to reversible, simultaneous suppression of homologous endogenous genes, producing variegated or white flowers.

Fire and Mello Experiment

The foundational RNAi experiment demonstrating that injecting a mixture of sense and antisense RNA (dsRNA) into C. elegans results in potent, targeted gene silencing compared to single strands alone.

Dicer

An endoribonuclease that cleaves long precursor double-stranded RNA (dsRNA) into short 21-to-23 nucleotide fragments known as small interfering RNA (siRNA).

RNA-Induced Silencing Complex (RISC)

A multiprotein effector complex comprising Dicer and Argonaute (AGO) that binds guide siRNA or miRNA strands to target, cleave, or translationally repress complementary mRNA sequences.

siRNA vs. miRNA

Two classes of small regulatory RNAs: siRNA is typically double-stranded, exogenous, or chemically synthesized, whereas miRNA is single-stranded, endogenous, and forms stem-loop precursors.

30-Nucleotide RNAi Limit

The maximum permissible nucleotide length for synthetic double-stranded RNA introduced into mammalian cells to trigger RNAi without inducing non-specific interferon immune responses.

Drosha

A nuclear RNase III enzyme that cleaves primary microRNA transcripts (pri-miRNAs) into shorter, individual precursor stem-loop structures (pre-miRNAs).

Seed Region

A conserved 7-to-8 nucleotide sequence located at the 5' end of a mature microRNA, essential for base-pairing and binding specificity to the 3' UTR of target mRNAs.

lin-4 and lin-14

A regulatory genetic circuit in C. elegans wherein lin-4 produces a non-coding microRNA that binds to the 3' UTR of lin-14 mRNA to control developmental timing and cell differentiation.

Patisiran

The first FDA-approved therapeutic drug utilizing RNA interference, designed to silence mutant and wild-type transthyretin mRNA in the liver to treat hereditary transthyretin amyloidosis.

Open Reading Frame (ORF)

A continuous sequence of nucleotide triplets in mRNA extending from an AUG initiation codon to a stop codon without interrupting punctuation or termination signals.

Degeneracy of the Genetic Code

The redundancy characteristic of the genetic code wherein multiple distinct codons specify the same amino acid residue.

Wobble Hypothesis

The principle proposed by Crick stating that non-Watson-Crick base pairing can occur at the third (3') position of an mRNA codon, frequently preventing amino acid substitution upon single-base mutation.

Polynucleotide Phosphorylase

A template-independent bacterial enzyme utilized by Marshall Nirenberg and Severo Ochoa to synthesize synthetic polyribonucleotide chains used to decipher the genetic code.

Stop Codons

The three terminal codons—UAA, UGA, and UAG—that direct the termination of protein translation by releasing the completed polypeptide from the ribosome.

Aminoacyl-tRNA Synthetases

Enzymes that attach specific amino acids to their cognate tRNA molecules via ester linkages to either the 2' hydroxyl (Class I) or 3' hydroxyl (Class II) group of the terminal adenosine.

Shine-Dalgarno Sequence

A conserved prokaryotic ribosomal binding sequence located upstream of the AUG start codon that base-pairs with 16S rRNA to properly position the 30S ribosomal subunit.

Kozak Scanning Model

The eukaryotic translation initiation model formulated by Marilyn Kozak, wherein the 40S subunit binds the 5' 7-methylguanosine cap and scans downstream to locate the initial AUG codon, promoted by a purine (A or G) at the -3 position.

Ribosomal Active Sites (A, P, E)

The three functional tRNA binding cavities inside an assembled ribosome: the Aminoacyl (A) site, Peptidyl (P) site, and Exit (E) site.

AlphaFold

An artificial intelligence system that computes and predicts three-dimensional protein structures with atomic accuracy directly from primary amino acid sequences.

Protein Phosphorylation

A reversible post-translational modification involving the enzymatic covalent addition of a phosphate group to the hydroxyl oxygen of serine, threonine, or tyrosine residues.

GSK-3 Consensus Motif

The target amino acid sequence required for phosphorylation by glycogen synthase kinase-3, defined by the sequence S/T-X-X-X-S/T.

Rous Sarcoma Virus (RSV)

An avian oncogenic retrovirus whose genome encodes four essential genes: gag (structural capsid), pol (reverse transcriptase), env (envelope glycoproteins), and src (transforming oncogene).

v-Src

A constitutively active cytoplasmic tyrosine kinase encoded by Rous Sarcoma Virus that causes continuous, dysregulated tyrosine phosphorylation and cellular oncogenic transformation.

Receptor Tyrosine Kinases (RTKs)

Cell-surface transmembrane receptors whose intracellular tyrosine kinase domain activates through autophosphorylation upon binding an extracellular regulatory ligand.

Philadelphia Chromosome

An aberrant truncated chromosome resulting from a reciprocal translocation fusing the BCR and ABL genes, producing an oncoprotein that drives chronic myelogenous leukemia (CML).

Gleevec (Imatinib)

A targeted small-molecule competitive kinase inhibitor specifically engineered to inhibit the constitutively active BCR-ABL tyrosine kinase oncoprotein in CML.

Pulse-Chase Experiment

A biochemical methodology measuring protein turnover rates by first pulse-labeling cells with radioactive amino acids and subsequently tracking signal decay during an unlabeled chase interval.

Ubiquitin (APF-1)

A highly conserved 76-amino acid regulatory polypeptide (originally identified as ATP-dependent proteolysis factor-1) that covalently conjugates to lysine residues on target proteins marked for degradation.

E1, E2, and E3 Enzymes

The sequential enzymatic triad of ubiquitination: E1 activates ubiquitin using ATP, E2 transfers activated ubiquitin, and E3 ligases provide target substrate recognition and specificity.

Lysine 48 Polyubiquitination

A specific polyubiquitin chain conformation formed via isopeptide bonds between the C-terminal diglycine of one ubiquitin and lysine 48 of the preceding ubiquitin, serving as the canonical signal for proteasomal destruction.

26S Proteasome

A large ($\sim 1500\,\text{kD}$) ATP-dependent cylindrical proteolysis machine consisting of a 20S catalytic core particle and two 19S regulatory particles (lid and base) that unfold and degrade tagged proteins.

Autophagy

A catabolic pathway whereby cytoplasmic contents, protein aggregates, or damaged organelles are sequestered into double-membraned autophagosomes and delivered to lysosomes for degradation, regulated by Apg12 and Apg8 complexes.

Luciferin and Luciferase

The two primary components of bioluminescence identified by Raphael Dubois, consisting of a light-emitting chemical substrate (luciferin) and its oxidative catalytic enzyme (luciferase).

Aequorin

A photoprotein isolated from Aequorea victoria that undergoes bioluminescence and emits blue light upon the binding of calcium ions ($Ca^{2+}$).

Green Fluorescent Protein (GFP)

A fluorescent protein natively emitting green light that functions without exogenous substrates, engineered by Roger Tsien with an S65T mutation (Ser65 to Thr) to significantly enhance its fluorescence output.

Stokes Shift

The spectral distance between the maximum wavelength of light absorbed and the maximum wavelength of light subsequently emitted by a fluorescent molecule.

Hybridoma Technology

A laboratory method created by Kohler and Milstein wherein antibody-secreting splenic B cells are fused with immortal myeloma cells to generate continuous monoclonal antibody clones.

Dual Luciferase Assay

A quantitative reporter gene system measuring luminescence sequentially from two different luciferases (experimental promoter versus constitutive internal control) to normalize transfection efficiency.

Somatic Cell Nuclear Transfer (SCNT)

A reproductive cloning procedure where the nucleus of a fully differentiated somatic cell is transferred into an enucleated oocyte, demonstrated in the cloning of Dolly the sheep.

Yamanaka Factors

The set of four transcription factors—Oct3/4, Sox2, Klf4, and c-Myc—capable of reprogramming mature, differentiated somatic cells back into induced pluripotent stem cells (iPSCs).

Teratoma Formation Assay

An in vivo validation test for pluripotency in which suspected stem cells are injected into immunocompromised mice to verify their ability to differentiate into tissues derived from all three embryonic germ layers.