Molecular Biology Exam 3 - Comprehensive Study Guide

Vocabulary practice flashcards covering lac and ara operon regulation, eukaryotic transcription, 5′ capping, pre-mRNA splicing, 3′-end processing, mRNA export, translation, turnover, and chromatin accessibility methods.

Operon

A group of bacterial structural genes transcribed together from shared regulatory sequences; the lac operon consists of lacZ, lacY, and lacA.

lacI / LacR

A separately transcribed gene encoding LacR, the lactose repressor. Without inducer, LacR binds lac operators to inhibit lacZYA transcription.

lacZ / β-galactosidase

lacZ encodes β-galactosidase (βgal), an enzyme that hydrolyzes lactose into glucose and galactose and converts a portion of lactose into the inducer allolactose.

lacY / LacY

lacY encodes lactose permease, a membrane transporter that imports lactose and other galactosides into the bacterial cell.

lacA

Encodes transacetylase; the third structural gene shown in the lac operon map.

lacP1 and lacP2

Two promoters for lacZYA. lacP1 is the primary, highly regulated promoter. lacP2 is a weaker promoter starting at approximately $-22$ relative to lacP1, with ~$200$-fold lower RNAP affinity and providing a ~$5$-fold basal boost under high glucose.

lacO1, lacO2, and lacO3

Three LacR-binding operator sites positioned at approximately $+11$, $+412$, and $-82$ relative to the principal transcription start site, respectively.

Allolactose

The physiological inducer of the lac operon; a β-1,6-linked lactose isomer formed by β-galactosidase that binds LacR, induces a conformational change, and reduces repressor affinity for operators.

LacR tetramer and repression loop

A V-shaped dimer-of-dimers that simultaneously binds two distant operator sites (especially O1 and O3), forming a DNA loop to enhance transcriptional repression.

Catabolite repression

The preferential utilization of glucose; low glucose triggers high cAMP and CRP/CAP-dependent transcriptional activation, whereas high glucose produces low cAMP and weak activation.

CRP / CAP

cAMP receptor protein (catabolite activator protein), a dimeric regulator that binds a symmetric DNA target when complexed with cAMP, bends DNA roughly $90^\circ$, and contacts the RNAP α-subunit to stimulate transcription across approximately $500$ genes.

IPTG

A nonmetabolized ("gratuitous") lactose inducer imported via LacY that binds LacR to alleviate repression, yielding ~$50\%$ induction at $2 \times 10^{-4}\,\text{M}$.

ONPG

A colorimetric substrate for β-galactosidase whose enzymatic cleavage produces a yellow-orange product measured by spectrophotometry; employed in screens and quantitative assays rather than selections.

X-gal

A chromogenic β-galactosidase substrate that yields blue colonies on agar plates; utilized for visual screening rather than survival-based selection.

TONPG

A toxic galactoside transported by LacY that acts as a killing or counterselection agent against bacteria constitutively expressing the lac operon.

lacI⁻ mutation

A loss-of-function mutation in the repressor gene that causes constitutive transcription of the lac operon (ON in the absence of inducer).

lacOᶜ mutation

An operator-constitutive cis-acting DNA mutation that prevents LacR binding, resulting in constitutive transcription of downstream linked lacZYA genes.

Equilibrium dialysis

An experimental technique where a radiolabeled ligand (such as [³H]-IPTG) equilibrates across a semipermeable membrane while a binding protein is retained; higher ligand concentration on the protein side indicates binding.

araBAD genes

Structural genes (araA, araB, and araD) encoding enzymes that metabolize L-arabinose through the pathway: L-arabinose $\rightarrow$ L-ribulose $\rightarrow$ L-ribulose-5-phosphate $\rightarrow$ L-xylulose-5-phosphate.

araC / AraC

Gene encoding a dimeric regulatory protein that represses the araBAD promoter in the absence of arabinose and activates it in the presence of arabinose.

araO2 and araI1

Two operator sites bound simultaneously by AraC in the absence of arabinose to establish a repressive DNA loop.

araI1 and araI2

Adjacent regulatory DNA sites occupied by AraC in the presence of arabinose, reconfiguring the protein to promote transcriptional activation of araP_BAD.

Helical phasing

The requirement that cooperative DNA-binding sites align on the same face of the DNA helix; insertions or deletions of half-turns (~$5$, $15$, or $25\,\text{bp}$) disrupt looping, whereas full turns (~$10$ or $31\,\text{bp}$) restore repression.

araE and araFGH

Arabinose transport genes located elsewhere in the bacterial genome and regulated by AraC; araE encodes a permease, and araFGH encodes a multicomponent uptake system.

RNA polymerase I

The eukaryotic RNA polymerase dedicated to transcribing the large precursor ribosomal RNA (pre-rRNA).

RNA polymerase II

The eukaryotic enzyme responsible for transcribing pre-mRNAs and several snRNAs; its largest subunit is designated Rpb1.

RNA polymerase III

The eukaryotic RNA polymerase that transcribes tRNAs, 5S rRNA, and select small functional RNAs.

Rpb1 CTD

The carboxy-terminal domain of RNA Polymerase II's largest subunit, functioning as an assembly platform to recruit 5′-capping, splicing, and 3′-end processing machineries during transcription.

5′ cap / Cap 0

A 7-methylguanosine residue linked via a distinctive 5′–5′ triphosphate bridge to the first nucleotide of eukaryotic pre-mRNA.

Cap 1 and Cap 2

Higher eukaryotic mRNA cap structures; Cap 1 features a 2′-O-methyl modification on the first transcribed nucleotide, while Cap 2 contains an additional 2′-O-methyl group on the second transcribed nucleotide.

Spliceosomal U snRNAs

U1, U2, U4, U5, and U6; the core small nuclear RNAs that assemble with proteins to execute pre-mRNA splicing (distinct from U3, U8, U13 in rRNA processing, and U7 in histone 3′-end formation).

5′ splice site

The 5′ exon–intron boundary, characterized by a conserved GU dinucleotide that is recognized early during spliceosome assembly through base pairing with U1 snRNA.

Branch point sequence (BPS)

A conserved intronic motif near the 3′ end containing a critical adenosine whose 2′-OH initiates splicing; yeast exhibits a strict UACUAAC sequence, whereas the human motif is more degenerate.

Polypyrimidine tract

A pyrimidine-rich tract located upstream of the 3′ splice site that facilitates recognition of the downstream intron boundary by splicing factors such as U2AF.

3′ splice site

The 3′ intron–exon boundary, typically marked by an AG dinucleotide, which serves as the substrate cleaved during the second catalytic step of splicing.

Sm ring

A conserved seven-membered ring composed of Sm proteins (D1, D2, F, E, G, D3, and B/B′) assembled on spliceosomal snRNAs.

SR proteins

Splicing regulatory proteins possessing N-terminal RNA-binding domains and C-terminal arginine/serine-rich (RS) domains that commonly promote spliceosome assembly.

Spliceosome ribozyme core

The catalytic active center formed by coordinated base-pairing interactions between U2 and U6 snRNAs and divalent $\text{Mg}^{2+}$ ions, specifically centered on nucleotides $59\text{--}82$ of U6 snRNA.

Transesterification 1

The first catalytic step of splicing in which the 2′-OH of the branch point adenosine attacks the 5′ splice site, generating a free 5′ exon and a branched lariat-intron intermediate.

Transesterification 2

The second catalytic step of splicing where the 3′-OH of the upstream exon attacks the 3′ splice site, ligating the two exons together and excising the intron as a lariat.

Lariat

The looped intron product generated during splicing that contains an unusual 2′–5′ phosphodiester linkage between the branch adenosine and the 5′ end of the intron.

Dscam alternative splicing

A Drosophila gene containing 24 exons with alternative cassettes ($12$ for exon 4, $48$ for exon 6, $33$ for exon 9, and $2$ for exon 17) capable of generating $12 \times 48 \times 33 \times 2 = 38{,}016$ distinct mRNA isoforms.

Exon skipping

A form of alternative splicing wherein a cassette exon is selectively retained in some transcript isoforms and entirely spliced out of others.

Mutually exclusive exons

An alternative splicing pattern where two consecutive alternative exons are regulated such that exactly one is included in the mature transcript, never both together.

Intron retention

A mode of alternative splicing in which a sequence typically excised as an intron is retained in the final mature mRNA.

ESE and ISE

Exonic splicing enhancers and intronic splicing enhancers; cis-regulatory RNA sequences that stimulate splice site utilization, often by recruiting SR proteins.

ESS and ISS

Exonic splicing silencers and intronic splicing silencers; cis-acting RNA motifs that repress splice site recognition, frequently by binding hnRNP factors.

hnRNP

Heterogeneous nuclear ribonucleoprotein; a family of RNA-binding proteins that mediate diverse RNA processing events and frequently act as splicing silencers.

RON epithelial vs ΔRON mesenchymal isoforms

Alternative splicing outcomes of the RON receptor; epithelial cells include exon 11 via SRSF2 binding to an ESE to produce regulated RON, whereas mesenchymal cells skip exon 11 to generate constitutively active ΔRON.

Back-splicing / exonic circular RNA

A noncanonical splicing pathway in which a downstream splice donor joins an upstream splice acceptor, excising an exon as a covalently closed circular RNA.

Microexon

A very short regulated exon (~$3\text{--}51\,\text{nt}$) highly conserved in vertebrates, enriched in neural, cardiac, and muscle tissues, that alters protein interaction surfaces without changing protein abundance.

TENM2 microexon

A ~$21\text{-nt}$ microexon whose alternative inclusion dictates latrophilin binding and modulates excitatory versus inhibitory synapse specificity.

USE / PAS / DSE

Upstream sequence element (often UGUA), polyadenylation signal (consensus AAUAAA), and downstream GU-rich element; three cis-elements that direct 3′-end cleavage and polyadenylation.

CPSF

Cleavage and polyadenylation specificity factor; a multisubunit complex that recognizes the AAUAAA PAS motif and harbors the CPSF73 cleavage endonuclease.

CSTF

Cleavage stimulation factor; a protein complex containing CSTF2/CSTF64 that binds the downstream GU-rich element (DSE) to stabilize the 3′-cleavage machinery.

CPSF73

The catalytic endonuclease within the CPSF complex that cleaves the pre-mRNA downstream of the PAS, typically at a CA dinucleotide.

PAP (Poly(A) polymerase)

The template-independent polymerase that adds adenosine residues to the newly cleaved 3′ end of eukaryotic mRNA.

Alternative cleavage/polyadenylation (APA)

The differential selection of cleavage/polyadenylation sites occurring in ~$68\%$ of human genes, which alters 3′ UTR length, switches terminal exons, or results in final exon skipping.

CSTF2 / NFATc1 regulation

A regulatory switch in T cells: low CSTF2 in naive cells results in distal PA2 selection, retaining the final exon, whereas elevated CSTF2 in effector cells directs proximal PA1 cleavage, producing a truncated isoform.

Torpedo termination

A transcription termination model where 3′ pre-mRNA cleavage leaves an uncapped nascent RNA end that is degraded by a 5′$\rightarrow$3′ exonuclease, which catches RNA Polymerase II to trigger release.

Promoter–terminator looping

A model in which physical interactions between initiation and termination factors bring the gene ends together, facilitating efficient recycling and reinitiation of RNA Polymerase II.

Exon junction complex (EJC)

A multiprotein complex deposited roughly $20\text{--}24\,\text{nt}$ upstream of spliced exon junctions that assists in nuclear export and mRNA surveillance.

NXF1/NXT1

The principal heterodimeric export receptor that binds mature mRNPs and mediates their translocation through nuclear pore complexes.

Nuclear pore complex (NPC)

A massive macromolecular structure embedded in the nuclear envelope comprising approximately $30$ nucleoporin types and hundreds of total protein subunits.

FG nucleoporins (FG-Nups)

Nucleoporins featuring phenylalanine–glycine repeats (such as FxFG and GLFG motifs) that form a selective hydrophobic meshwork interacting with export receptors.

eIF4F

The translation initiation complex that recognizes the 5′ cap; composed of eIF4E (cap-binding subunit), eIF4A (RNA helicase), and eIF4G (scaffolding factor).

IRES

Internal ribosome entry site; an RNA structural element that recruits ribosomes internally without relying on the canonical 5′ cap, often used in engineered polycistronic vectors.

Deadenylation

The progressive shortening of the poly(A) tail at the 3′ end of mRNA, usually acting as the initial rate-limiting event preceding decapping and transcript turnover.

Terminal nucleotidyl transferases (TENTs)

A family of $11$ mammalian template-independent RNA tailing enzymes, certain members of which catalyze 3′ polyuridylation to accelerate mRNA decay.

RNA editing

Post-transcriptional base modification mechanisms, including adenosine-to-inosine deamination (A$\rightarrow$I, read as G) and cytidine-to-uridine deamination (C$\rightarrow$U).

N⁶-methyladenosine (m⁶A)

The most prevalent internal mRNA chemical modification (~$3$ marks per transcript) that influences translation, RNA decay, nuclear export, and alternative splicing.

DNase I hypersensitive site (DHS)

A chromatin region unusually vulnerable to cleavage by DNase I due to the absence of canonical nucleosomes and the occupancy of transcription factors.

DNase I hypersensitivity assay

A Southern blot-based assay in which isolated nuclei are treated with DNase I, genomic DNA is isolated and digested with restriction enzymes, and fragments are probed to detect accessible genomic loci.

ATAC-seq

Assay for transposase-accessible chromatin with sequencing; uses hyperactive Tn5 transposase to simultaneously cut and tag open chromatin regions with sequencing adapters.