Rho factor: ATP-dependent translocase in prokaryotes.
A: Initiation = Sigma factor role ✗
B: Elongation = Core RNA polymerase ✗
C: Termination of transcription = Rho factor ✓
D: Post-transcriptional modification = eukaryotic (capping, tailing, splicing) ✗
Answer: C — Termination of transcription
RNA polymerase: core enzyme (alpha2, beta, beta-prime, omega) + sigma factor = holoenzyme. Sigma factor: recognises promoter (-10 and -35 boxes). Most common: sigma-70 in E. coli for housekeeping genes. Other sigma factors for stress responses (sigma-32 for heat shock, sigma-38 for stationary phase). Promoter: -35 box (TTGACA) and -10 box (Pribnow box: TATAAT). Consensus sequences. Binding to promoter: holoenzyme binds double-stranded DNA (closed complex). Melting of DNA around -10 box (open complex). RNA synthesis begins at +1. Sigma dissociates after ~10 nucleotides.
Rho-independent (intrinsic) termination: inverted repeat in DNA → GC-rich hairpin in mRNA transcript. RNA pol pauses at hairpin. Followed by run of U residues. Weak rU:dA base pairs in the RNA:DNA hybrid melt easily → RNA transcript released → transcription ends. No protein factors needed. Rho-dependent termination: Rho protein: hexameric ring-shaped ATP-dependent RNA translocase. Binds rut (Rho utilisation) site on mRNA: cytosine-rich, unstructured region. Translocates 5 to 3 along mRNA. When RNA pol pauses (at termination site with poor template), Rho catches up. Rho unwinds RNA:DNA hybrid using ATP hydrolysis → mRNA released → termination. Both pathways: RNA pol pausing is key. After termination, RNA pol, DNA, and transcript separate. Rho factor also involved in anti-sense RNA regulation and transcription-translation coupling.
Jacob and Monod (1961): operon model for gene regulation in E. coli. Lac operon: structural genes (lacZ=beta-galactosidase, lacY=permease, lacA=transacetylase) under single promoter. Operator: DNA sequence between promoter and structural genes where repressor binds. Repressor (encoded by lacI): binds operator → blocks RNA pol → transcription OFF. When lactose present: allolactose (isomer of lactose) binds repressor → conformational change → repressor cannot bind operator → transcription ON. Catabolite repression: CAP (catabolite activator protein) + cAMP activates lac operon. High glucose → low cAMP → CAP inactive → lac operon off (even if lactose present). Inducible vs repressible operons: Lac = inducible (substrate = inducer). Trp operon = repressible (end product = co-repressor activates repressor).
Three RNA polymerases: Pol I: nucleolus, transcribes rRNA genes (28S, 18S, 5.8S). Pol II: nucleoplasm, transcribes protein-coding genes (mRNA), snRNA, miRNA. Pol III: tRNA, 5S rRNA, snRNA U6. General transcription factors (GTFs): TFIID binds TATA box (recognises promoter). TFIIH: helicase (unwinds DNA), kinase (phosphorylates Pol II CTD). Preinitiation complex (PIC): Pol II + TFIIA, B, D, E, F, H assembled at promoter. Enhancers: can be thousands of bp from gene. DNA loops bring enhancer to promoter region. Mediator complex: transmits enhancer signals to Pol II. Silencers: reduce transcription. Insulators: block enhancer-promoter communication.
5-prime capping: 7-methylguanosine (m7GTP) added to 5-end co-transcriptionally. Functions: protects mRNA from 5-exonuclease, promotes ribosome binding (cap-dependent translation). 3-prime polyadenylation: cleavage at poly-A signal (AAUAAA) by CPSF. Poly-A polymerase adds ~200 adenosines. Functions: mRNA stability, nuclear export, translation efficiency. Splicing: introns removed, exons joined by spliceosome. Spliceosome: snRNPs (U1, U2, U4, U5, U6). GT-AG rule: introns start with GU, end with AG. Branch point A: 2-OH attacks 5-splice site → lariat intermediate. Exons join. Alternative splicing: same pre-mRNA → different mRNAs → different proteins. Human proteome diversity largely from alternative splicing (~100,000+ proteins from ~20,000 genes). mRNA export: through nuclear pore complexes after processing. Surveillance: NMD (nonsense-mediated decay) degrades mRNAs with premature stop codons.
miRNA (microRNA): small ~22 nt ncRNAs. Encoded in genome. Processed by Drosha (nucleus) then Dicer (cytoplasm) into mature miRNA. Loaded into RISC complex. Complementary to 3-UTR of target mRNA → translational repression or mRNA degradation. ~2000 human miRNAs regulate >50% of protein-coding genes. Key roles in development, cell differentiation, cancer. siRNA (small interfering RNA): similar to miRNA but from long double-stranded RNA. RNAi (RNA interference) = gene silencing mechanism. Used experimentally for gene knockdown. Therapeutic: siRNA drugs approved (patisiran for hereditary transthyretin amyloidosis). lncRNA (long non-coding RNA): >200 nt. Thousands in human genome. Functions: chromatin remodelling (Xist: X-chromosome inactivation), transcriptional regulation, scaffolding. XIST lncRNA: coats inactive X chromosome, recruits PRC2 complex for H3K27me3 (repressive histone mark). piRNA (Piwi-interacting RNA): in germ cells. Silences transposons. Protects genome integrity.
Rifampicin: antibiotic. Binds bacterial RNA polymerase beta subunit (active site channel). Blocks extension of short (<3 nt) RNA transcripts. Does not inhibit eukaryotic RNA pol. Major antibiotic for TB (Mycobacterium tuberculosis). Also used for meningococcal prophylaxis. Resistance: mutations in rpoB gene (encodes beta subunit). Alpha-amanitin: toxin from Amanita phalloides (death cap mushroom). Potent inhibitor of eukaryotic RNA Pol II (at very low concentrations). Also inhibits Pol III at higher concentrations. Does not inhibit Pol I or prokaryotic RNA pol. Mechanism: binds between bridge helix and trigger loop of Pol II, prevents translocation. Actinomycin D: intercalates into DNA (especially GC-rich regions). Blocks RNA pol translocation. Inhibits both transcription and DNA replication. Used in cancer chemotherapy (Wilms tumour, rhabdomyosarcoma).
Riboswitches: RNA elements in the 5-UTR of mRNA that change conformation based on small molecule binding. Regulate transcription (terminator hairpin formation) or translation. Metabolite binds riboswitch → structural change → gene expression changes. Examples: thiamine riboswitch (B1 vitamin - controls thiamine biosynthesis genes), SAM riboswitch (S-adenosylmethionine), lysine riboswitch, guanidine riboswitches. Transcriptional attenuation (trp operon): leader sequence before trpE structural gene. During translation: if tryptophan abundant, ribosome translates leader quickly (Trp residues in leader), adopts terminator hairpin → transcription terminates. If tryptophan scarce, ribosome stalls at Trp codons, anti-terminator forms → transcription continues through structural genes. Elegant coupled transcription-translation regulation system unique to prokaryotes.