Molecular Biology Exam 2 Study Guide: RNA Processing, Translation, and Non-Coding RNAs

Comprehensive vocabulary flashcards covering molecular biology techniques, co-transcriptional RNA processing, splicing mechanisms, tRNA charging and decoding, ribosome structure, translation cycle stages, stress response pathways, and small regulatory RNAs (miRNAs/siRNAs).

Northern Blot

A molecular biology technique that detects specific RNA size and abundance through RNA gel electrophoresis, membrane transfer, and hybridization with a labeled complementary DNA probe.

Western Blot

An analytical technique used to detect specific protein size and abundance by separating proteins via SDS-PAGE, transferring them to a membrane, and probing with a primary antibody followed by a labeled secondary antibody.

Ribo-seq (Ribosome Profiling)

A high-resolution technique that maps ribosome positions on mRNAs during active translation by freezing ribosomes with drugs, digesting unprotected RNA with nuclease, and sequencing the remaining ~30 nt protected ribosome footprints.

RNA Interference (RNAi)

A biological mechanism and reverse-genetics method to assess gene function through loss of function by introducing exogenous dsRNA or siRNA complementary to target mRNA, triggering target cleavage via Dicer and RISC/Argonaute.

Reporter Construct

An experimental construct that tests the activity of a regulatory element (such as a promoter, enhancer, or UTR) by fusing it to an easily quantifiable reporter gene, such as lacZ, GFP, or luciferase.

RNA-seq

A sequencing technique that converts all cellular RNAs into cDNA and sequences them to analyze total transcriptome expression levels and identify exon-junction reads demonstrating alternative splice isoforms.

RT-PCR (Reverse Transcription PCR)

A targeted technique that detects specific RNA transcripts by reverse transcribing RNA into cDNA followed by PCR amplification with gene-specific primers, revealing product size differences on a gel for alternative isoforms.

ChIP-seq (Chromatin Immunoprecipitation Sequencing)

A method used to detect genome-wide protein–DNA binding sites and histone modifications (such as H3K9me3) by crosslinking, shearing chromatin, immunoprecipitating with a specific antibody, and sequencing the bound DNA.

EMSA (Electrophoretic Mobility Shift Assay)

An in vitro technique that detects protein–nucleic acid binding by observing the retarded electrophoretic migration (shifted band) of a labeled nucleic acid probe complexed with protein on a native gel.

RNA Polymerase II C-Terminal Domain (CTD)

A flexible protein tail consisting of heptapeptide repeats on RNA Polymerase II that acts as a scaffold; its reversible phosphorylation recruits 5' capping enzymes, spliceosomal machinery, and 3' polyadenylation factors co-transcriptionally.

Ser5 Phosphorylation (Ser5-P)

The phosphorylation of serine 5 on the RNA Polymerase II CTD during transcription initiation and early elongation that recruits capping enzymes to add the 5' m7G cap within ~20–30 nt of transcription.

Ser2 Phosphorylation (Ser2-P)

The phosphorylation of serine 2 on the RNA Polymerase II CTD during transcription elongation that acts alongside Ser5-P to recruit spliceosome components, and persists near termination to recruit cleavage and polyadenylation factors CPSF and CstF.

5' Cap (m7G Cap)

A modified 7-methylguanosine cap linked via a reverse 5'–5' triphosphate bridge to the nascent mRNA 5' end that protects mRNA from 5'→3' exonucleases, promotes nuclear export, and is bound by eIF4E for translation initiation.

snRNPs (Small Nuclear Ribonucleoproteins)

RNA-protein complexes (U1, U2, U4, U5, U6) that form the core of the spliceosome, utilizing their small nuclear RNA components to recognize splice junctions via base-pairing with pre-mRNA (e.g., U1 pairs with the 5' splice site; U2 pairs with the branchpoint).

Branchpoint Adenosine

A conserved adenine nucleotide located within an intron whose 2'-OH group launches a nucleophilic attack on the 5' splice site in step 1 of splicing, generating a characteristic lariat intermediate.

CPSF and CstF

Cleavage and polyadenylation factors recruited by the phosphorylated CTD; CPSF recognizes and binds the upstream AAUAAA signal, while CstF binds a downstream GU/U-rich element to direct transcript cleavage ~10–30 nt downstream.

Poly(A) Polymerase (PAP)

A template-independent polymerase that synthesizes a polyadenine tail of approximately 200 adenosine residues onto the newly cleaved 3' end of pre-mRNA.

Exon Skipping (Cassette Exon)

An alternative splicing event in which a cassette exon is spliced out together with its flanking introns, omitting the entire exon from the mature transcript.

Intron Retention

A splicing variation in which an intron fails to be excised and is retained within the mature mRNA, increasing transcript size and frequently introducing in-frame premature termination codons.

Cryptic Splice Site

A dormant or non-canonical splice sequence that resembles a donor (GU) or acceptor (AG) site and becomes active upon mutation or loss of normal sites, resulting in exonic lengthening or shortening.

Splicing Enhancers and Silencers (ESEs, ISEs, ESSs, ISSs)

Cis-acting exonic and intronic sequence motifs that bind activator or repressor proteins to stimulate or inhibit spliceosome assembly at adjacent splice sites.

Aminoacyl-tRNA Synthetase (aaRS)

One of 20 family enzymes responsible for charging cognate tRNAs by catalyzing a two-step reaction: activating an amino acid with ATP into aminoacyl-AMP, then transferring the amino acid to the 3' CCA terminal adenosine of the tRNA.

Double Sieve Mechanism

A proofreading system in aminoacyl-tRNA synthetases where an activation site excludes amino acids that are too large, while an editing site hydrolytically removes smaller or isosteric near-miss amino acids that mistakenly fit into the activation site.

Wobble Hypothesis

The rule governing non-standard base-pairing between the 5' base of a tRNA anticodon and the 3' base of an mRNA codon, enabling a single tRNA species to decode multiple degenerate codons.

Inosine (I)

A post-transcriptionally deaminated adenosine base found at the 5' wobble position of certain tRNAs that can base-pair with U, C, or A at the 3' position of an mRNA codon.

Nucleolus

A non-membrane-bound nuclear compartment where Pol I transcribes 45S pre-rRNA, snoRNPs mediate covalent base modifications and cleavage, and ribosomal proteins assemble with rRNAs into 40S and 60S subunits.

snoRNPs (Small Nucleolar Ribonucleoproteins)

Nuclear complexes consisting of small nucleolar RNAs and proteins that base-pair with 45S pre-rRNA to direct site-specific modifications (such as 2'-O-methylation and pseudouridylation) and targeted endonucleolytic processing.

Peptidyl Transferase Center (PTC)

The catalytic center of the large ribosomal subunit (60S in eukaryotes, 50S in prokaryotes) composed entirely of rRNA, demonstrating that the ribosome functions as a ribozyme to catalyze peptide bond formation.

A Site (Aminoacyl Site)

The ribosomal site that receives incoming cognate aminoacyl-tRNAs delivered by elongation factors, or binds release factors when a stop codon enters the decoding center.

P Site (Peptidyl Site)

The ribosomal site that carries the peptidyl-tRNA attached to the growing nascent polypeptide chain, and specifically accommodates initiator Met-tRNAi during translation initiation.

E Site (Exit Site)

The ribosomal site that transiently holds deacylated tRNA molecules following peptidyl transfer and translocation before they are ejected from the ribosome.

eIF4F Complex (eIF4E, eIF4G, eIF4A)

The translation initiation complex comprising eIF4E (which binds the 5' m7G cap), eIF4A (an ATP-dependent RNA helicase that unwinds 5' UTR secondary structures), and eIF4G (a scaffold protein bridging eIF4E and PABP to circularize the mRNA).

eIF2

A regulatory GTPase initiation factor that binds GTP and initiator Met-tRNAi to form the ternary complex required for 40S small subunit scanning and AUG start codon selection.

eEF1A (Bacterial EF-Tu)

A GTPase elongation factor that delivers aminoacyl-tRNAs to the ribosomal A site and governs kinetic proofreading via GTP hydrolysis timing upon correct codon–anticodon matching.

eEF2 (Bacterial EF-G)

A translation elongation GTPase that catalyzes the coordinated translocation of the ribosome by three nucleotides along the mRNA toward the 3' end, shifting tRNAs from A and P sites to P and E sites.

Release Factors (e.g., eRF1)

Proteins mimicking tRNA structures that recognize mRNA stop codons (UAA, UAG, UGA) in the ribosomal A site and activate the peptidyl transferase center to hydrolyze the peptidyl-tRNA ester bond.

Kinetic Proofreading (Translation)

A fidelity mechanism during translation elongation where correct codon–anticodon base pairing accelerates EF1 GTP hydrolysis and peptide bond formation, while mismatches prolong GTP hydrolysis and allow dissociation of incorrect aa-tRNAs.

Open Reading Frame (ORF)

A continuous stretch of mRNA codons beginning with an AUG start codon, consisting of an integer multiple of 3 nucleotides, and terminating at an in-frame stop codon without internal stops.

Integrated Stress Response (ISR)

A cellular adaptive pathway in which stress kinases (GCN2, PERK, PKR, HRI) phosphorylate eIF2α, sequestering eIF2B to suppress global cap-dependent translation initiation while preferentially upregulating stress-remedying transcription factors like ATF4.

Upstream Open Reading Frame (uORF)

A small reading frame located within the 5' UTR of an mRNA that acts as a cis-regulatory element to repress downstream main ORF translation under non-stress conditions.

ATF4 (Activating Transcription Factor 4)

A mammalian transcriptional activator whose translation increases during integrated stress through delayed reinitiation past inhibitory uORFs, activating amino acid synthesis, transport genes, and the GADD34 recovery phosphatase.

Exon Junction Complex (EJC)

A multiprotein complex deposited ~20–24 nt upstream of exon–exon junctions upon pre-mRNA splicing that is cleared by the pioneer round of ribosome translation and serves as a positional marker for nonsense-mediated decay.

Nonsense-Mediated Decay (NMD)

A surveillance pathway that eliminates aberrant mRNAs containing premature termination codons upstream of an exon junction complex (EJC), recognized by Upf proteins to trigger rapid RNA degradation.

MicroRNA (miRNA)

An endogenous ~22 nt non-coding regulatory RNA derived from imperfect hairpin precursors that guides miRISC to 3' UTR sequences of target mRNAs via seed pairing, leading to translational repression and exonucleolytic decay.

Small Interfering RNA (siRNA)

A ~21–22 nt double-stranded RNA molecule derived from long, perfectly base-paired dsRNA precursors that directs RISC to cleave target mRNAs endonucleolytically or establish transcriptional heterochromatin.

miRNA Seed Region

The conserved 5' sequence of a microRNA (spanning nucleotides 2–7 or 2–8) that requires precise, uninterrupted Watson-Crick base-pairing with mRNA 3' UTR target sites to anchor the miRISC complex.

Drosha and DGCR8 (Microprocessor)

A nuclear RNase III enzyme complex that crops primary miRNA (pri-miRNA) transcripts into ~70 nt precursor miRNA (pre-miRNA) hairpin intermediates.

Exportin-5

A nuclear export receptor that recognizes the short stem and 2-nucleotide 3' overhang of pre-miRNA hairpins to transport them through nuclear pore complexes to the cytoplasm.

Dicer

A cytoplasmic RNase III endoribonuclease that cleaves the loop from pre-miRNA hairpins and processes long dsRNA into ~21–22 nt mature small RNA duplexes.

Argonaute (Ago / Ago2)

The core catalytic protein component of RISC and miRISC that binds the small RNA guide strand and uses its PIWI domain to execute endonucleolytic slicing or recruit silencing effectors.

RITS (RNA-Induced Transcriptional Silencing)

A nuclear Argonaute-containing complex in fission yeast that utilizes siRNAs against centromeric dh repeats to target nascent transcripts and recruit H3K9 methyltransferases, establishing stable heterochromatin.