Nucleic Acid Replication, Transcription, Translation, and Regulatory Mechanisms

Nucleic Acid Replication, Transcription, Translation, and Regulatory Mechanisms

I. Central Dogma of Molecular Biology:

• Overview: Describes the flow of genetic information within a biological system.
• DNA Replication: DNA makes a copy of itself. Ensures genetic information is passed to daughter cells during cell division.  
• Transcription: DNA sequence is transcribed into RNA (specifically mRNA, tRNA, rRNA). Converts genetic information from DNA form to RNA form.  
• Translation: mRNA sequence is translated into a protein sequence. Decodes RNA information to synthesize proteins, the functional molecules of the cell.  

 

• Simplified Flow: DNA → RNA → Protein

II. DNA Replication:

• Definition: The process of producing two identical copies of DNA from one original DNA molecule. Essential for cell division and inheritance.  
• Basic Principles:
• Semi-conservative: Each new DNA molecule consists of one original (template) strand and one newly synthesized strand.  
• Template-dependent: The sequence of the new strand is determined by the sequence of the template strand, following base-pairing rules (A-T, G-C).  
• Direction of Synthesis: DNA polymerase always adds nucleotides to the 3' end of a growing strand. Thus, DNA synthesis proceeds in the 5' to 3' direction.  

A. Replication in Prokaryotes (e.g., E. coli)

• Key Players:
• Origin of Replication (oriC): Specific DNA sequence where replication initiates. E. coli has a single oriC.  
• Initiator Proteins (DnaA): Recognize and bind to oriC, causing local unwinding of DNA.  
• Helicase (DnaB): Unwinds the double helix at the replication fork, separating the two strands.  
• Single-Stranded Binding Proteins (SSBPs): Bind to single-stranded DNA to prevent strands from re-annealing and protect them from nucleases.  
• Primase (DnaG): Synthesizes short RNA primers to provide a 3'-OH group for DNA polymerase to begin synthesis.  
• DNA Polymerase III: The main replication enzyme in E. coli. Highly processive and accurate. Has 5'→3' polymerase and 3'→5' exonuclease (proofreading) activity.  
• DNA Polymerase I: Removes RNA primers and replaces them with DNA. Also has 5'→3' exonuclease activity.  
• DNA Ligase: Seals the nicks (phosphodiester bond gaps) between DNA fragments.  
• Topoisomerases (Gyrase): Relieve torsional stress ahead of the replication fork caused by unwinding.  
• Steps of Replication:

1.               Initiation:

• Initiator proteins bind to oriC.  
• Helicase is recruited and unwinds DNA.  
• SSBPs stabilize single strands.  
• Primase synthesizes RNA primers.  

2.               Elongation:

• DNA Polymerase III extends primers, adding dNTPs to the 3'-OH end.  
• Leading Strand: Synthesized continuously in the 5'→3' direction towards the replication fork, requiring only one initial primer.
• Lagging Strand: Synthesized discontinuously in short fragments (Okazaki fragments) in the 5'→3' direction away from the replication fork. Each Okazaki fragment requires a new RNA primer.

3.               Termination:

• Replication forks meet at the ter sites (termination sequences) on the circular chromosome.  
• Termination proteins block further replication fork progression.  
• Topoisomerases separate the intertwined daughter DNA molecules (decatenation).  
• DNA Polymerase I removes RNA primers and fills gaps.  
• DNA ligase seals nicks.  

• Regulation in Prokaryotes:
• Initiation Control: Replication is primarily regulated at the initiation step.
• DnaA-ATP levels: DnaA protein is active when bound to ATP. Levels of DnaA-ATP are regulated.  
• oriC Methylation: oriC region contains GATC sequences that are methylated on adenine bases by Dam methylase. Newly replicated DNA is hemimethylated (only parental strand methylated). Hemimethylated oriC is transiently inactive, preventing immediate re-initiation. Full methylation is required for efficient initiation.  
• SeqA protein: Binds to hemimethylated oriC and inhibits premature re-initiation. Result [1] indicates SeqA is involved in high-affinity binding of hemimethylated oriC. Result [1] also notes that oriC remains hemimethylated for about 30-40% of the cell cycle. This delay ensures replication initiation is coordinated with cell cycle.  
• Ratios of ATP to ADP bound to DnaA: High ATP promotes initiation.  
• Availability of resources (dNTPs, energy).
• Replication Licensing: Ensures each origin fires only once per cell cycle. Hemimethylation and SeqA binding contribute to this.

B. Replication in Eukaryotes (e.g., Human Cells)

• Key Players:
• Multiple Origins of Replication: Eukaryotic chromosomes are much larger and linear, so they have multiple origins of replication to speed up replication.  
• Origin Recognition Complex (ORC): Binds to origins throughout the cell cycle.  
• MCM Helicase: The main helicase in eukaryotes, loaded onto DNA origins in early G1 phase (licensing).  
• Cdc6 and Cdt1: Proteins required for loading MCM helicase onto origins during licensing, inactivated after initiation to prevent re-licensing.  
• Cyclin-Dependent Kinases (CDKs) and DDK: Kinases that activate origins to initiate replication in S phase. Phosphorylate proteins like MCM helicase to activate them and trigger origin firing.  
• Replication Protein A (RPA): Eukaryotic SSBPs.
• DNA Polymerase α: Primase activity and initiates DNA synthesis (but has no proofreading).
• DNA Polymerase ε: Primarily replicates the leading strand. High processivity and proofreading activity.
• DNA Polymerase δ: Primarily replicates the lagging strand. High processivity and proofreading activity.
• FEN1 (Flap Endonuclease 1) and RNase H: Remove RNA primers (FEN1 is a flap endonuclease).  
• DNA Ligase I: Seals nicks in DNA.  
• Topoisomerases I and II: Relieve torsional stress.  
• Steps of Replication: Similar to prokaryotes (Initiation, Elongation, Termination), but with key differences:

1.               Initiation:

• ORC binds to origins.  
• Licensing: MCM helicases are loaded onto origins in G1.  
• Origin activation in S phase: CDKs and DDK activate licensed origins.  
• Helicases unwind DNA.  
• RPA stabilizes single strands.  
• DNA Polymerase α with primase activity initiates synthesis.

2.               Elongation:

• DNA Polymerase ε (leading) and δ (lagging) extend DNA strands.
• Leading and lagging strand synthesis similar to prokaryotes with Okazaki fragments on lagging strand.

3.               Termination:

• Replication forks meet and fuse.  
• Topoisomerases decatenate daughter DNA molecules.  
• Primer removal by FEN1 and RNase H, gap filling, and ligation.  
• Telomere replication: Special mechanisms (telomerase) are required to replicate the ends of linear chromosomes to prevent shortening with each replication cycle.  

• Regulation in Eukaryotes:
• Origin Licensing and Activation: Strict control to ensure each origin fires only once per cell cycle. Licensing in G1 and activation in S phase (CDK and DDK dependent).
• Cell Cycle Control: Replication is tightly linked to the cell cycle. Initiation occurs only in S phase. CDKs play a key role in both cell cycle progression and replication initiation.  
• Checkpoint Mechanisms: DNA damage checkpoints monitor replication and cell cycle progression, arresting the cycle if problems are detected (e.g., DNA damage, stalled replication forks).  

III. Transcription:

• Definition: The process of synthesizing an RNA molecule from a DNA template.  
• Types of RNA produced: mRNA (messenger RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), and various regulatory RNAs (miRNA, siRNA, etc.). mRNA is the template for protein synthesis.  
• Basic Principles:
• Template-dependent: RNA sequence is complementary to the DNA template strand.  
• Direction of Synthesis: RNA polymerase synthesizes RNA in the 5' to 3' direction, using a DNA template read in the 3' to 5' direction.  
• Base Pairing: Similar to DNA replication, but uracil (U) replaces thymine (T) in RNA (A-U, G-C pairing).
• Promoters: DNA sequences that signal the start of a gene and where RNA polymerase binds.

A. Transcription in Prokaryotes (E. coli)

• Key Players:
• RNA Polymerase: A single RNA polymerase complex is responsible for transcribing all types of RNA (mRNA, tRNA, rRNA).
• Core Enzyme: Catalytic activity (α₂ββ'ω subunits). Can synthesize RNA but cannot initiate specifically.  
• Sigma (σ) Factor: A subunit that associates with the core enzyme for promoter recognition and initiation specificity. Different sigma factors recognize different promoter sequences, allowing for regulation of gene expression. (e.g., σ⁷⁰ for most genes, σ³² for heat shock genes).  
• Promoters: DNA sequences upstream of genes that signal transcription start. E. coli promoters have two main consensus sequences:
• -10 region (Pribnow box): TATAAT consensus sequence, located ~10 bases upstream of the transcription start site (+1).  
• -35 region: TTGACA consensus sequence, located ~35 bases upstream of the +1 site.

 

• Terminator Sequences: DNA sequences that signal the end of transcription. Can be rho-dependent or rho-independent.  
• Steps of Transcription:

1.               Initiation:

• Sigma factor associates with RNA polymerase core enzyme to form the holoenzyme.  
• Holoenzyme binds to the promoter region. Sigma factor recognizes and binds to the -10 and -35 promoter elements.  
• RNA polymerase unwinds DNA around the initiation site to form a transcription bubble.  
• RNA polymerase starts synthesizing RNA at the +1 site, after initiation, sigma factor is released.

2.               Elongation:

• RNA polymerase moves along the DNA template, unwinding DNA ahead and rewinding DNA behind the transcription bubble.  
• RNA polymerase synthesizes RNA in the 5'→3' direction, adding rNTPs to the 3'-OH end, complementary to the template strand.  

3.               Termination:

• Rho-independent (Intrinsic) Termination: Terminator sequence forms a hairpin loop structure in the RNA transcript, followed by a string of Us. The hairpin destabilizes the RNA-DNA interaction, and weak U-A base pairing causes RNA polymerase to dissociate and transcription to stop.  
• Rho-dependent Termination: Rho (ρ) factor is a protein that binds to a specific sequence on the RNA transcript (rut site) and moves along the RNA towards RNA polymerase. When RNA polymerase pauses at a terminator sequence, Rho factor catches up and uses its helicase activity to unwind the RNA-DNA hybrid, causing termination.  

• Regulation in Prokaryotes:
• Sigma Factors: Different sigma factors direct RNA polymerase to different sets of promoters, allowing for global regulation of gene expression (e.g., heat shock response, sporulation).  
• Promoter Strength: Efficiency of transcription initiation is influenced by the similarity of the promoter sequence to the consensus sequence. Stronger promoters have sequences closer to the consensus and are transcribed more efficiently.  
• Transcription Factors (Activators and Repressors): Proteins that bind to DNA near promoters and can either stimulate (activators) or inhibit (repressors) transcription initiation. Often respond to environmental signals.
• Operons: Sets of genes transcribed together from a single promoter and regulated coordinately. Common in prokaryotes.
• Inducible Operons (e.g., lac operon): Transcription is normally off and is induced (turned on) in the presence of an inducer molecule (e.g., lactose).  
• Repressible Operons (e.g., trp operon): Transcription is normally on and is repressed (turned off) in the presence of a corepressor molecule (e.g., tryptophan).  

 

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• Attenuation: A regulatory mechanism that controls transcription elongation based on the availability of the end product (e.g., tryptophan in trp operon).  

B. Transcription in Eukaryotes (e.g., Human Cells)

• Key Players:
• Three RNA Polymerases:
• RNA Polymerase I: Transcribes rRNA genes (except 5S rRNA). Located in the nucleolus.
• RNA Polymerase II: Transcribes mRNA precursors (pre-mRNA), snRNA genes (involved in splicing), and miRNA genes. Located in the nucleoplasm. This is the main RNA polymerase for gene expression.
• RNA Polymerase III: Transcribes tRNA genes, 5S rRNA gene, and other small RNAs. Located in the nucleoplasm.
• General Transcription Factors (GTFs): Proteins required for basal transcription at all promoters transcribed by each RNA polymerase. For RNA Polymerase II, these include TFIIB, TFIID (TBP subunit recognizes TATA box), TFIIE, TFIIF, TFIIH.  
• Promoters: More diverse and complex than prokaryotic promoters. For RNA Polymerase II, common elements include:
• TATA box: ~ -25 bp upstream of start site (consensus TATAAAA). Binding site for TBP (TATA-binding protein), a subunit of TFIID.  
• Initiator element (Inr): Around the transcription start site.  
• CpG islands: GC-rich regions often found in promoters of housekeeping genes.  
• Upstream regulatory sequences: CAAT box, GC box, etc.  

 

• Enhancers and Silencers: Regulatory DNA sequences that can be located far away from the promoter (upstream or downstream, even within introns). Enhancers stimulate transcription, silencers inhibit transcription. Act through mediator proteins and chromatin modifications.  
• Mediator Complex: Large multiprotein complex that mediates interaction between transcription factors bound to enhancers/silencers and the RNA polymerase II complex at the promoter.  
• Chromatin Structure: DNA is packaged into chromatin in eukaryotes. Chromatin structure (histone modifications, DNA methylation) plays a major role in regulating transcription accessibility.  
• Steps of Transcription: More complex than prokaryotes. Focus on RNA Polymerase II transcription (mRNA synthesis).

1.               Initiation:

• GTFs assemble at the promoter region to form the pre-initiation complex (PIC). TFIID (TBP) binds to TATA box. TFIIB, TFIIF, RNA Pol II, TFIIE, and TFIIH are sequentially recruited.  
• TFIIH has helicase activity to unwind DNA at the start site and kinase activity to phosphorylate RNA Pol II CTD (C-terminal domain), which is crucial for promoter clearance and elongation.
• Formation of transcription bubble and transcription initiation.

2.               Elongation:

• RNA Polymerase II moves along the template, synthesizing RNA.
• Elongation factors assist RNA polymerase II in efficient and processive transcription.  
• Transcription coupled to RNA processing: As pre-mRNA emerges from RNA Pol II, it undergoes processing (capping, splicing, polyadenylation).  

3.               Termination:

• Less well-defined termination signals than in prokaryotes.
• For protein-coding genes, termination is often linked to polyadenylation. After the polyadenylation signal (AAUAAA) is transcribed, the RNA transcript is cleaved downstream, and poly(A) tail is added at the 3' end. Termination may be coupled to cleavage.  

• RNA Processing in Eukaryotes (Post-transcriptional modifications): Pre-mRNA needs to be processed in the nucleus to become mature mRNA before translation.  
• 5' Capping: Addition of a 7-methylguanosine cap to the 5' end of the pre-mRNA. Important for mRNA stability, translation initiation, and splicing.  
• 3' Polyadenylation: Addition of a poly(A) tail (string of adenine nucleotides) to the 3' end. Important for mRNA stability, translation, and termination.
• RNA Splicing: Removal of introns (non-coding sequences) and joining of exons (coding sequences) in pre-mRNA. Carried out by the spliceosome, a complex of snRNPs (small nuclear ribonucleoproteins).
• Alternative Splicing: Splicing can occur in different ways, leading to different mRNA isoforms from the same gene, increasing protein diversity.  

 

• Regulation in Eukaryotes: Highly complex and multi-layered.  
• Chromatin Remodeling: Changes in chromatin structure (e.g., histone acetylation, DNA methylation) can control gene accessibility to transcription machinery. Euchromatin (relaxed) is generally transcriptionally active; heterochromatin (condensed) is inactive.  
• Transcription Factors (Activators and Repressors): A large number of transcription factors bind to promoters and enhancers/silencers to regulate transcription initiation. Combinatorial control: gene expression is regulated by the specific combination of transcription factors present.  
• Enhancers and Silencers: Long-range regulatory elements that can control gene expression from a distance.  
• Coactivators and Corepressors: Proteins that do not bind DNA directly but interact with transcription factors to enhance or repress transcription. Mediator complex is a key coactivator.  
• RNA Processing Control: Regulation at steps of 5' capping, splicing, 3' polyadenylation, and mRNA stability can influence gene expression levels. Alternative splicing is a major regulatory mechanism for generating protein diversity.  
• mRNA Stability and Degradation: mRNA lifespan is regulated and influences the amount of protein produced. Factors like poly(A) tail length, 5' cap, and RNA-binding proteins affect mRNA stability.  
• Non-coding RNAs (ncRNAs): miRNAs, siRNAs, lncRNAs (long non-coding RNAs) play diverse regulatory roles in transcription, RNA processing, and translation. For example, miRNAs can regulate gene expression by binding to mRNA and causing translational repression or mRNA degradation.  

IV. Translation:

• Definition: The process of synthesizing a polypeptide (protein) chain using the information encoded in mRNA.
• Cellular Machinery: Ribosomes, tRNA, mRNA, and various protein factors.
• Genetic Code: Set of rules by which information encoded in genetic material (DNA or RNA sequences) is translated into proteins.
• Codon: A triplet of nucleotides in mRNA that specifies an amino acid or a stop signal.  
• 64 Codons: 61 codons specify 20 amino acids (degeneracy/redundancy of the code). 3 stop codons (UAA, UAG, UGA) signal termination.  
• Start Codon: AUG (methionine) signals the start of translation.  
• Universal Code (mostly): The genetic code is largely universal across all organisms, with minor variations.  

 

A. Translation in Prokaryotes (E. coli)

• Key Players:
• Ribosomes (70S): Composed of two subunits: 30S (small subunit) and 50S (large subunit). rRNA (16S rRNA in 30S, 23S and 5S rRNA in 50S) and proteins. Ribosomes catalyze peptide bond formation.
• A site (aminoacyl-tRNA site): Binds incoming aminoacyl-tRNA.
• P site (peptidyl-tRNA site): Holds tRNA with the growing polypeptide chain.  
• E site (exit site): Site where empty tRNA exits the ribosome.  

 

• tRNA (transfer RNA): Adapter molecules that bring specific amino acids to the ribosome based on the mRNA codon. Each tRNA has an anticodon loop that base-pairs with the mRNA codon and an amino acid attachment site at the 3' end where the corresponding amino acid is attached (aminoacylated tRNA or charged tRNA).  
• mRNA (messenger RNA): Contains the coding sequence (codons) for the protein. Prokaryotic mRNAs are often polycistronic (encode multiple genes in one mRNA molecule).  
• Initiation Factors (IF1, IF2, IF3): Assist in ribosome assembly and initiation.  
• Elongation Factors (EF-Tu, EF-G, EF-Ts): Facilitate tRNA binding, peptide bond formation, and ribosome translocation during elongation.  
• Release Factors (RF1, RF2, RF3): Recognize stop codons and trigger termination.  
• Steps of Translation:

1.               Initiation:

• 30S ribosomal subunit binds to mRNA. Shine-Dalgarno sequence (ribosome-binding site) in mRNA upstream of start codon helps in ribosome binding and positioning.  
• Initiator tRNA (fMet-tRNA^fMet^) carrying formylmethionine (fMet) binds to the start codon (AUG) in the P site. IF2-GTP mediates initiator tRNA binding. IF1 and IF3 block A and E sites.  
• 50S ribosomal subunit joins to form the 70S initiation complex. IFs are released, and GTP is hydrolyzed to GDP.  

 

2.               Elongation: Cycle of three steps:

• Codon Recognition: Aminoacyl-tRNA with the correct anticodon for the next mRNA codon (in the A site) binds to the A site. EF-Tu-GTP delivers tRNA to the A site. GTP hydrolysis and EF-Tu-GDP release occur after correct codon-anticodon interaction.  
• Peptide Bond Formation: Peptidyl transferase activity of the 23S rRNA (in 50S subunit) catalyzes peptide bond formation between the amino acid in the A site and the growing polypeptide chain in the P site. Peptide chain is transferred from tRNA in P site to tRNA in A site.  
• Translocation: Ribosome moves one codon down the mRNA in the 5' direction. EF-G-GTP is required. tRNA in the P site moves to the E site (and exits), tRNA in the A site (now with the polypeptide chain) moves to the P site, and the A site becomes vacant for the next aminoacyl-tRNA. GTP hydrolysis accompanies translocation.  

3.               Termination:

• When a stop codon (UAA, UAG, or UGA) enters the A site, there is no tRNA that recognizes it.  
• Release factors (RF1 or RF2, depending on the stop codon) recognize the stop codon and bind to the A site.  
• RF3-GTP facilitates RF1/RF2 binding.
• Release factors promote hydrolysis of the bond between the polypeptide chain and tRNA in the P site, releasing the polypeptide.  
• Ribosome subunits dissociate, mRNA and tRNA are released. Ribosome recycling factor (RRF) and EF-G help in ribosome dissociation.  

• Regulation in Prokaryotes:
• Transcriptional Control (major): Primarily regulated at the level of transcription initiation (operons, sigma factors, transcription factors).  
• Translational Control (minor, but important in some cases):
• mRNA Secondary Structure: Secondary structures in the 5' untranslated region (5'UTR) of mRNA can block ribosome binding or scanning and inhibit translation initiation.  
• Antisense RNA: Small RNA molecules complementary to mRNA can bind to mRNA and block ribosome binding or promote mRNA degradation.  
• Riboswitches: mRNA sequences that can bind small molecules and change their conformation, affecting translation initiation or termination.  
• Protein Factors: Some proteins can bind to mRNA and regulate its translation.  
• Codon Usage: Frequency of codon usage can influence translation rate. Rare codons can slow down ribosome movement.  

B. Translation in Eukaryotes (e.g., Human Cells)

• Key Players:
• Ribosomes (80S): Larger than prokaryotic ribosomes. Composed of 40S (small subunit) and 60S (large subunit). rRNA and proteins.
• tRNA: Similar to prokaryotic tRNA, but initiator tRNA carries methionine (Met-tRNA^Met^), not formylmethionine.
• mRNA: Eukaryotic mRNAs are monocistronic (encode only one protein per mRNA). Have 5' cap and 3' poly(A) tail, which enhance translation.  
• Eukaryotic Initiation Factors (eIFs): More complex set of initiation factors (eIF1, eIF2, eIF3, eIF4F, eIF5, eIF6, etc.). eIF4F is a complex that includes eIF4E (cap-binding protein), eIF4G (scaffold protein), and eIF4A (helicase).  
• Eukaryotic Elongation Factors (eEF1A, eEF2): Similar function to prokaryotic elongation factors, but different proteins.
• Eukaryotic Release Factors (eRF1, eRF3): Release factors.  
• Steps of Translation:

1.               Initiation: More complex and highly regulated than in prokaryotes.

• 40S ribosomal subunit, with eIFs and initiator tRNA-Met (Met-tRNA^Met^) bound, associates with mRNA. eIF4F complex (with eIF4E binding to 5' cap) and eIF4G interact with mRNA.  
• 40S subunit scans mRNA from the 5' cap in the 5'→3' direction to find the start codon (AUG) in a favorable sequence context (Kozak sequence). ATP-dependent scanning.  
• Once start codon is found, initiator tRNA base-pairs with AUG codon. GTP hydrolysis by eIF2.  
• 60S ribosomal subunit joins to form the 80S initiation complex. eIFs are released.  

2.               Elongation: Similar to prokaryotic elongation (codon recognition, peptide bond formation, translocation), but uses eukaryotic elongation factors (eEF1A, eEF2).  

3.               Termination: Similar to prokaryotic termination, but uses eukaryotic release factors (eRF1 and eRF3). eRF1 recognizes all three stop codons. eRF3-GTP promotes ribosome release.  

• Regulation in Eukaryotes: Highly regulated at multiple levels.  
• Transcriptional Control (major): Chromatin structure, transcription factors, enhancers, etc.
• RNA Processing Control: Splicing, alternative splicing, mRNA stability.
• Translational Control (important):
• Initiation is the key regulatory step: eIF4F complex and eIF2 are major targets for regulation.  
• Phosphorylation of eIF2α: Phosphorylation of eIF2α (by kinases activated by stress, nutrient deprivation, viral infection) inhibits eIF2B, reducing eIF2-GTP regeneration and thus reducing translation initiation. A general mechanism to globally reduce translation under stress conditions.  
• Regulation of eIF4F complex: Activity of eIF4F is regulated. 4E-BPs (eIF4E-binding proteins) can bind to eIF4E and inhibit eIF4F complex assembly. Phosphorylation of 4E-BPs (by mTOR kinase in response to growth factors and nutrients) releases eIF4E, promoting translation initiation.  
• 5' UTR sequences and structures: Secondary structures, upstream open reading frames (uORFs) in the 5'UTR can regulate translation.  
• miRNA regulation: miRNAs bind to the 3'UTR of target mRNAs and repress translation or promote mRNA degradation. A major mechanism for gene regulation.  
• mRNA Localization: Localization of mRNA to specific cellular regions can regulate translation in space and time.  
• Post-translational Control: Protein folding, modification, trafficking, and degradation are also important levels of gene regulation.

 

V. Summary Table: Prokaryotes vs. Eukaryotes

Feature	Prokaryotes	Eukaryotes
DNA Replication	Single origin (oriC), circular DNA	Multiple origins, linear chromosomes
Initiation Protein	DnaA	ORC, multiple licensing factors (MCM, Cdc6, Cdt1)
Main DNA Polymerase	DNA Pol III	DNA Pol ε (leading), Pol δ (lagging)
Origins/chromosome	One	Multiple
Transcription	Single RNA Polymerase	Three RNA Polymerases (I, II, III)
RNA Polymerase	Core enzyme + sigma factor	RNA Pol II requires GTFs (TFIIB, TFIID, etc.)
Promoter	-10 and -35 boxes, simple	TATA box, Inr, CpG islands, complex, enhancers
RNA Processing	Minimal (no nucleus)	Extensive (5' cap, splicing, 3' poly(A) tail)
mRNA structure	Polycistronic, Shine-Dalgarno sequence	Monocistronic, 5' cap, poly(A) tail, Kozak sequence
Translation	70S ribosomes	80S ribosomes
Initiation tRNA	fMet-tRNA^fMet^	Met-tRNA^Met^
Coupling	Transcription and translation are coupled	Transcription and translation are spatially and temporally separated (nucleus vs. cytoplasm)
Regulation	Operons, sigma factors, transcription factors	Chromatin remodeling, transcription factors, enhancers, RNA processing, translation control, miRNAs