Source: Comprehensive Guide to DNA Recombination, Gene Expression, and Regulation (UCF)
Tags: central dogma, transcription, RNA polymerase, sigma factor, promoter, -10 box, -35 box, Pribnow box, open complex, closed complex, promoter escape, abortive initiation, elongation, termination, rho-dependent, rho-independent, intrinsic termination, eukaryotic transcription, RNA Pol I, RNA Pol II, RNA Pol III, TFIID, TBP, TFIIH, CTD, PIC, preinitiation complex, Mediator complex, 5' capping, polyadenylation, splicing, spliceosome, snRNP, alternative splicing, RNA editing, tRNA, translation, ribosome, Shine-Dalgarno, Kozak, EF-Tu, EF-G, antibiotics
Difficulty: Intermediate to Advanced Prerequisites: Central dogma basics (DNA to RNA to protein), DNA structure, and an introductory understanding of enzyme function. Part 1 of these notes (DNA Recombination and Repair) is helpful but not strictly required for this section.
Gene expression begins with transcription (copying DNA into RNA) and, for protein-coding genes, continues through RNA processing and translation (decoding mRNA into protein). These are the processes through which the information stored in DNA is converted into the molecules that do the cell's work. Bacteria and eukaryotes share the same core logic but differ substantially in the details, particularly in promoter architecture, RNA processing requirements, and the complexity of the regulatory machinery. This set of notes walks through transcription, RNA processing, and translation in both systems, along with the antibiotics that target each step.
RNA polymerase transcribes DNA into RNA in three phases (initiation, elongation, termination). Eukaryotic mRNAs are extensively processed (capping, splicing, polyadenylation) before export to the cytoplasm. Translation decodes mRNA into protein at the ribosome, using tRNAs as adaptors. Bacteria and eukaryotes use mechanistically similar but distinct machinery at each step, and several antibiotics exploit those differences.
Central dogma
The flow of genetic information: DNA → RNA → Protein. DNA stores the instructions; RNA serves as messenger and functional molecule; proteins execute cellular tasks.
RNA polymerase (RNAP)
The multi-subunit enzyme that synthesises RNA from a DNA template, working 5' to 3'. Described as having a "crab claw" shape. Uses Mg2+ for catalysis and lacks the 3'→5' exonuclease proofreading of DNA polymerases.
Sigma factor (σ)
A dissociable subunit of bacterial RNA polymerase holoenzyme that directs the core enzyme to specific promoter sequences. Different sigma factors (e.g., σ70, σ54) recognise different promoter motifs, enabling gene regulation in response to environmental cues.
-35 box
A conserved hexanucleotide sequence (~TTGACA) located approximately 35 bases upstream of the transcription start site in bacterial promoters. Recognised by the sigma factor.
-10 box (Pribnow box)
A conserved AT-rich hexanucleotide sequence (~TATAAT) located approximately 10 bases upstream of the start site in bacterial promoters. The site where DNA strand separation (melting) begins.
Closed complex
The initial complex in which RNA polymerase holoenzyme is bound to the promoter but the DNA remains double-stranded.
Open complex
The complex formed after DNA melts (unwinds) in the region from approximately -11 to +3, exposing the template strand for RNA synthesis.
Promoter escape
The transition from initiation to productive elongation, during which the polymerase clears the promoter, releases the sigma factor, and begins synthesising full-length RNA.
Abortive initiation
The repeated synthesis and release of short RNA transcripts (fewer than ~10 nt) before the polymerase successfully escapes the promoter.
Intrinsic (rho-independent) termination
Transcription termination in bacteria triggered by a GC-rich RNA hairpin followed by a run of uracils (poly-U). The hairpin destabilises the elongation complex and the weak rU-dA base pairs allow dissociation.
Rho-dependent termination
Transcription termination in bacteria mediated by the Rho helicase, which binds C-rich rut (rho utilisation) sites on the nascent RNA, translocates along the transcript, and unwinds the RNA-DNA hybrid to release the polymerase.
General transcription factors (GTFs)
Proteins required for basal transcription from eukaryotic Pol II promoters: TFIID (contains TBP), TFIIB, TFIIF, TFIIE, TFIIH. They assemble in an ordered sequence to form the preinitiation complex.
TATA-binding protein (TBP)
A subunit of TFIID that recognises and binds the TATA box in eukaryotic promoters, nucleating PIC assembly.
Preinitiation complex (PIC)
The full assembly of GTFs and RNA Pol II at a eukaryotic promoter, poised to begin transcription.
C-terminal domain (CTD)
A repetitive heptapeptide tail on the largest subunit of RNA Pol II. Phosphorylation of its serine residues (especially Ser5 and Ser2) by kinases in TFIIH and P-TEFb regulates the transition from initiation to elongation and couples transcription with RNA processing.
5' cap
A 7-methylguanosine added to the 5' end of eukaryotic mRNA shortly after transcription begins. Protects the transcript from degradation and is required for ribosome recognition during translation.
Polyadenylation
The addition of a poly(A) tail (typically 100-250 adenines) to the 3' end of eukaryotic mRNA after cleavage at a polyadenylation signal. Stabilises the mRNA and aids nuclear export.
Splicing
The removal of introns and joining of exons in eukaryotic pre-mRNA, carried out by the spliceosome. Produces mature mRNA.
Spliceosome
A large ribonucleoprotein complex composed of five snRNPs (U1, U2, U4, U5, U6) plus associated proteins. Recognises splice sites via RNA-RNA base pairing and catalyses intron removal through two sequential transesterification reactions.
Alternative splicing
The inclusion or exclusion of different exons from the same pre-mRNA, generating multiple mRNA (and therefore protein) isoforms from a single gene. A major source of proteomic diversity.
RNA editing
Post-transcriptional modification of individual nucleotides in mRNA. Two main types: C-to-U (by cytidine deaminases) and A-to-I (by ADAR enzymes). Can alter codons and gene expression.
Shine-Dalgarno sequence (RBS)
A purine-rich sequence upstream of the start codon in bacterial mRNA that base-pairs with the 3' end of 16S rRNA, positioning the ribosome for translation initiation.
Kozak sequence
The consensus sequence surrounding the start AUG codon in eukaryotic mRNA (approximately GCCRCCAUGG). Recognised during ribosome scanning.
tRNA (transfer RNA)
Small RNA molecules (~75-94 nt) that serve as adaptors between mRNA codons and amino acids. Charged by aminoacyl-tRNA synthetases (aaRS). Feature a cloverleaf secondary structure with an anticodon loop and an acceptor stem.
P bodies (processing bodies)
Cytoplasmic granules where mRNAs are stored, silenced, or degraded. Contain decapping enzymes, exonucleases, and components of the mRNA decay machinery.
DNA → RNA → Protein
DNA: the stable repository of genetic information
RNA: messenger (mRNA), functional (rRNA, tRNA, regulatory RNAs)
Protein: the functional output that carries out most cellular processes
Aspect | Replication | Transcription |
|---|---|---|
Purpose | Copy the entire genome | Synthesise specific RNA segments |
Template | Both DNA strands (whole genome) | One strand of a gene (template/antisense) |
Primer required | Yes | No |
Fidelity | Very high (~1 error per 10^7 bases) | Lower (~1 error per 10^4 bases) |
Products | DNA strands | RNA molecules |
Timing | Once per cell cycle | Multiple times, on demand |
Multi-subunit enzyme; "crab claw" shape encloses the active site channel
Synthesises RNA 5' to 3' using the 3' to 5' DNA template strand
Lacks 3'→5' exonuclease proofreading (unlike DNA polymerase)
Uses two Mg2+ ions for catalysis (two-metal-ion mechanism)
RNA sequence produced is complementary to the template strand and identical to the coding (sense) strand (except U replaces T)
1. Initiation
RNA polymerase (with sigma factor in bacteria, or GTFs in eukaryotes) binds the promoter
DNA melts (unwinds) around the start site to form the open complex
First phosphodiester bonds are formed
2. Elongation
Polymerase moves along the template, synthesising RNA
The transcription bubble travels with the enzyme
Supercoiling ahead and behind the bubble is managed by topoisomerases
3. Termination
The completed transcript and enzyme are released
Mechanisms differ between bacteria and eukaryotes (see below)
-35 box (~TTGACA): initial recognition site for the sigma factor
-10 box / Pribnow box (~TATAAT): AT-rich region where strand melting initiates
UP-element: A/T-rich sequence upstream of -35 that enhances RNAP binding by interacting with the alpha subunit CTD
Discriminator: sequence between -10 and +1 that influences open complex stability
Spacing between the -35 and -10 boxes is critical (optimally ~17 bp)
σ70 (RpoD): the primary ("housekeeping") sigma factor in E. coli
σ54 (RpoN): activated by enhancer-binding proteins; used for nitrogen metabolism and other specialised functions
σ32 (RpoH): heat-shock response
σS (RpoS): stationary phase and stress response
Swapping sigma factors allows global reprogramming of gene expression
Closed complex: RNAP holoenzyme binds the promoter; DNA still fully double-stranded
Open complex: DNA melts from -11 to +3, exposing the template; ready for NTP binding
Abortive initiation: polymerase repeatedly synthesises and releases short transcripts (<10 nt) while still bound to the promoter
Promoter escape: conformational changes in RNAP displace sigma factor contacts with the promoter, allowing the enzyme to clear the promoter and enter productive elongation
RNAP moves at 50-90 nt/sec
Maintains a ~12-14 bp transcription bubble
Proofreading occurs by two mechanisms:
Pyrophosphorolytic editing: reversal of the polymerisation reaction to remove the last nucleotide
Hydrolytic editing: RNAP backtracks, and Gre factors (GreA/GreB) stimulate cleavage of the extruded RNA, allowing the polymerase to resume
Intrinsic (rho-independent):
A GC-rich palindromic sequence in the RNA folds into a stable hairpin
Followed by a run of U residues (poly-U)
The hairpin destabilises the elongation complex; weak rU-dA base pairs allow dissociation
Rho-dependent:
The Rho protein (a hexameric helicase) binds C-rich rut (rho utilisation) sites on the nascent RNA
Rho translocates 5'→3' along the RNA, catches up with the paused polymerase, and unwinds the RNA-DNA hybrid
Rifamycin (e.g., rifampicin): binds near the active site channel, blocking extension of RNA chains beyond 2-3 nt. Used clinically against tuberculosis.
Streptolydigin: inhibits conformational changes needed for the translocation step
Actinomycin D: intercalates into DNA, physically blocking RNAP progression
Resistance mechanisms: point mutations in RNAP, decreased membrane permeability, methylation of the drug target
RNA Pol I: synthesises most rRNAs (18S, 28S, 5.8S). Located in the nucleolus. Requires SL1 and UBF.
RNA Pol II: synthesises mRNA and most snRNAs. The main focus for gene regulation. Has the CTD.
RNA Pol III: synthesises tRNAs, 5S rRNA, and other small RNAs. Requires TFIIIA, TFIIIB, TFIIIC. Some promoters are internal to the transcribed region.
Pol IV and Pol V (plants only): produce small interfering RNAs (siRNAs) involved in RNA-directed DNA methylation
Requires assembly of the preinitiation complex (PIC) at the promoter:
TFIID (TBP subunit) binds the TATA box
TFIIB binds and helps position Pol II
TFIIF escorts Pol II to the promoter
TFIIE and TFIIH join; TFIIH has helicase and kinase activities
Promoter elements: TATA box, INR (initiator), DPE (downstream promoter element), DCE (downstream core element)
Chromatin remodelling: nucleosome modifications (histone acetylation opens chromatin; methylation can open or close it) regulate whether the promoter is accessible
Enhancers and UAS: distant regulatory sequences that bind activator proteins, often communicating with the promoter via DNA looping
TFIIH phosphorylates Ser5 of the CTD, triggering promoter escape
GTFs are released; elongation factors are recruited
Mediator complex: a large multi-subunit complex that bridges transcription activators and Pol II, stimulating transcription
Elongation factors:
FACT: disassembles nucleosomes ahead of Pol II and reassembles them behind
TFIIS: rescues stalled/backtracked polymerases by stimulating RNA cleavage
SPT5 and P-TEFb: promote processivity; P-TEFb phosphorylates Ser2 of the CTD and releases Pol II from promoter-proximal pausing
Coupled to 3' end processing (cleavage and polyadenylation)
Torpedo model: after cleavage at the poly(A) signal, a 5'→3' exonuclease (Rat1/Xrn2) degrades the downstream RNA still associated with Pol II; when it catches the polymerase, the complex disassembles
Allosteric model: passage through the poly(A) signal triggers a conformational change in Pol II that destabilises the elongation complex
5' capping: a 7-methylguanosine is added to the 5' end of the nascent mRNA co-transcriptionally. Protects against degradation, aids ribosome recruitment, and is required for efficient splicing of the first intron.
Splicing: introns are removed and exons joined by the spliceosome through two transesterification steps. The branch-point adenosine attacks the 5' splice site (forming a lariat), then the free 3' OH of exon 1 attacks the 3' splice site, joining the exons.
Polyadenylation: after cleavage at the AAUAAA signal, poly(A) polymerase adds 100-250 A residues. Stabilises the mRNA and promotes nuclear export.
Alternative splicing: different combinations of exons are included or excluded, producing multiple protein isoforms from one gene. Regulated by SR proteins (promote exon inclusion) and hnRNPs (promote exon skipping). Errors in splicing (exon skipping, use of pseudo splice sites) can cause disease, e.g., Duchenne muscular dystrophy.
Post-transcriptional base modifications that alter the coding sequence:
C-to-U editing: by cytidine deaminases (e.g., APOBEC1 editing of apolipoprotein B mRNA)
A-to-I editing: by ADAR enzymes; inosine is read as guanosine by the ribosome
Particularly important in the nervous system, where A-to-I editing of glutamate receptor mRNAs alters channel properties
Mature mRNA is exported through nuclear pores, mediated by exportins and the Ran GTPase cycle
mRNA stability is controlled by:
3' poly(A) tail length: deadenylation is often the first step in mRNA decay
5' cap removal (decapping): exposes the mRNA to 5'→3' exonucleases
AU-rich elements (AREs) in the 3' UTR: recruit destabilising factors
Nonsense-mediated decay (NMD): degrades mRNAs with premature stop codons, a quality-control mechanism
P bodies: cytoplasmic granules where mRNAs are stored or degraded
~75-94 nucleotides; cloverleaf secondary structure with four stem-loops
Key features: acceptor stem (3' CCA end where the amino acid is attached), anticodon loop, D loop, TΨC loop
Aminoacyl-tRNA synthetases (aaRS) charge each tRNA with its correct amino acid, using identity elements (specific bases and structural features) for recognition
Post-transcriptional modifications (methylation, pseudouridine, inosine) are critical for stability and accurate codon-anticodon pairing
Bacteria:
Shine-Dalgarno sequence on the mRNA base-pairs with the 3' end of 16S rRNA
Positions the start codon (AUG) in the ribosomal P-site
Initiator tRNA: fMet-tRNAfMet
Initiation factors: IF1, IF2, IF3
30S subunit binds mRNA and initiator tRNA, then 50S joins to form 70S
Eukaryotes:
The 5' cap is recognised by eIF4E (part of the eIF4F complex, with eIF4G and eIF4A helicase)
40S subunit, loaded with eIFs and Met-tRNAiMet, scans from the 5' cap to the first AUG in a favourable Kozak context
60S subunit joins to form 80S
Initiator tRNA: Met-tRNAiMet (not formylated)
Aminoacyl-tRNA delivery: EF-Tu (bacteria) or eEF1A (eukaryotes) delivers charged tRNA to the ribosomal A-site in a GTP-dependent step
Peptide bond formation: catalysed by the peptidyl transferase centre of the large subunit, which is a ribozyme (the catalytic activity comes from rRNA, not protein)
Translocation: EF-G (bacteria) or eEF2 (eukaryotes) promotes movement of the ribosome one codon along the mRNA, shifting tRNAs from A→P and P→E sites
Codon-anticodon pairing fidelity: geometry of the decoding centre in the small subunit (rRNA) checks for correct base-pairing
GTP hydrolysis by EF-Tu: provides a kinetic proofreading step; incorrect tRNAs dissociate before GTP is hydrolysed
Hydrolytic editing: if an incorrect amino acid is attached to a tRNA, some aaRS enzymes have an editing site that removes it
Stop codons (UAG, UGA, UAA) are recognised by release factors (RF1/RF2 in bacteria, eRF1 in eukaryotes), not by tRNAs
Release factors trigger hydrolysis of the peptidyl-tRNA bond, freeing the completed polypeptide
Ribosome recycling factor (RRF) and EF-G dissociate the ribosomal subunits from the mRNA, allowing them to participate in new rounds of translation
Puromycin: structural mimic of aminoacyl-tRNA; enters the A-site, forms a peptide bond, but causes premature chain release because it cannot translocate. Affects both bacterial and eukaryotic ribosomes.
Fusidic acid: inhibits EF-G, blocking translocation
Chloramphenicol: inhibits peptidyl transferase on bacterial 50S
Tetracycline: blocks aminoacyl-tRNA binding to the bacterial 30S A-site
Erythromycin: blocks the exit tunnel of the bacterial 50S subunit
These antibiotics exploit structural differences between bacterial (70S) and eukaryotic (80S) ribosomes
Proteins may be phosphorylated, methylated, acetylated, ubiquitinated, or lipid-modified after translation
Signal sequences (N-terminal hydrophobic stretches) direct proteins to the ER, mitochondria, or other organelles; cleaved after targeting
Nuclear localisation signals (NLS): short basic amino acid sequences that direct import into the nucleus via importin proteins
Poly(A)-binding proteins (PABP) interact with eIF4G at the 5' end, bringing the 3' and 5' ends of the mRNA together into a closed-loop structure
This enhances translation reinitiation by recycling ribosomes from the stop codon back to the start codon
Rifampicin (a rifamycin) is a front-line drug for tuberculosis treatment, targeting bacterial RNA polymerase. Understanding splicing has clinical importance: therapeutic antisense oligonucleotides (e.g., nusinersen for spinal muscular atrophy) work by redirecting alternative splicing. Many antibiotics used clinically (tetracycline, erythromycin, chloramphenicol) target bacterial ribosomes, making the structural differences between 70S and 80S ribosomes medically significant.
Students often think RNA polymerase reads the coding strand. It reads the template (antisense) strand 3' to 5', and the RNA produced matches the coding strand sequence.
The -10 and -35 boxes are sometimes confused with eukaryotic promoter elements. These are bacterial. Eukaryotic Pol II promoters use the TATA box, INR, DPE, etc.
Students sometimes say "the ribosome makes peptide bonds." More precisely, the peptidyl transferase activity resides in the rRNA of the large subunit (it is a ribozyme), not in ribosomal proteins.
Abortive initiation is often mistaken for a failed or pathological event. It is a normal part of initiation; most promoters produce several abortive transcripts before the polymerase escapes.
⚠️ Know the three phases of transcription and what happens at each.
⚠️ Be able to distinguish the two bacterial termination mechanisms (intrinsic vs. rho-dependent).
⚠️ Understand the order of PIC assembly: TFIID → TFIIB → Pol II/TFIIF → TFIIE → TFIIH.
⚠️ Know the three eukaryotic RNA processing steps (capping, splicing, polyadenylation) and when each occurs.
⚠️ The spliceosome's two transesterification steps are commonly tested.
⚠️ Understand the difference between bacterial and eukaryotic translation initiation (Shine-Dalgarno vs. cap-dependent scanning).
⚠️ Know which antibiotics target which step and which ribosomal subunit.
True or false: RNA polymerase requires a primer to begin transcription.
Fill in the blank: The bacterial promoter element where DNA melting begins is the __________.
True or false: TFIIH has both helicase and kinase activities.
Fill in the blank: In bacterial translation initiation, the __________ sequence on the mRNA base-pairs with 16S rRNA.
True or false: Peptide bond formation is catalysed by ribosomal proteins.
Answers: 1. False. 2. -10 box (Pribnow box). 3. True. 4. Shine-Dalgarno. 5. False (catalysed by rRNA, a ribozyme).
Q: Compare intrinsic and rho-dependent transcription termination in bacteria.
A: Intrinsic termination relies on sequences within the RNA itself: a GC-rich palindrome that folds into a hairpin, followed by a poly-U tract. The hairpin destabilises the elongation complex, and the weak rU-dA base pairs allow the transcript to dissociate. Rho-dependent termination requires the Rho helicase, which binds C-rich rut sites on the nascent RNA, translocates along the transcript using ATPase activity, and unwinds the RNA-DNA hybrid at the polymerase, causing release.
Q: Describe the assembly of the preinitiation complex at a Pol II promoter.
A: Assembly begins when TFIID (via its TBP subunit) binds the TATA box. TFIIB then binds, creating a platform for Pol II, which is escorted to the promoter by TFIIF. TFIIE and TFIIH join last. TFIIH uses its helicase activity to melt the DNA around the start site and its kinase activity to phosphorylate Ser5 of the CTD, triggering promoter escape.
Q: What are the three major co-transcriptional and post-transcriptional processing steps for eukaryotic mRNA, and what is the function of each?
A: (1) 5' capping: a 7-methylguanosine cap is added to the 5' end, protecting the mRNA from degradation and facilitating ribosome recruitment. (2) Splicing: introns are removed and exons joined by the spliceosome, producing a continuous coding sequence. (3) 3' polyadenylation: a poly(A) tail is added after cleavage at the polyadenylation signal, stabilising the mRNA and aiding nuclear export.
Q: How does translation initiation differ between bacteria and eukaryotes?
A: In bacteria, the Shine-Dalgarno sequence upstream of the start codon base-pairs with the 16S rRNA of the 30S subunit, positioning the AUG directly at the P-site. The initiator tRNA is fMet-tRNAfMet. In eukaryotes, the 40S subunit is recruited to the 5' cap via eIF4F, loads Met-tRNAiMet, and scans along the 5' UTR until it finds the first AUG in a favourable Kozak context. There is no Shine-Dalgarno equivalent.
Q: Why is the peptidyl transferase centre considered a ribozyme?
A: Structural and biochemical evidence shows that the catalytic activity of the peptidyl transferase centre resides in the 23S rRNA (in bacteria) rather than in the ribosomal proteins. The rRNA positions the substrates and stabilises the transition state for peptide bond formation. This makes it a ribozyme: an RNA molecule with enzymatic activity.
Transcription is the primary point of gene regulation (covered in Part 4 of these notes), where activators, repressors, and chromatin modifications control whether and how often a gene is transcribed. Splicing connects to alternative splicing regulation in eukaryotic gene expression. Translation fidelity and ribosome function also connect back to the antibiotics that are clinically important in microbiology and infectious disease.
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