Transcription, Translation, Gene Expression, and Regulation in Prokaryotes and Eukaryotes

Transcription, Translation, and Gene Expression: Overview

Central Dogma of Molecular Biology

The central dogma describes the flow of genetic information from DNA to RNA to protein. This process involves two main steps: transcription (DNA to RNA) and translation (RNA to protein). Gene expression refers to the entire process by which information from a gene is used to synthesize a functional gene product, typically a protein.

• Transcription: Synthesis of RNA from a DNA template by RNA polymerase.

• Translation: Synthesis of a polypeptide (protein) from an mRNA template by ribosomes.

• Gene Expression Regulation: Control of the timing, location, and amount of gene product produced.

• Relationship: Transcription produces mRNA, which is then translated into protein; regulation can occur at multiple steps.

• Example: The lac operon in Escherichia coli is regulated at the transcriptional level in response to lactose availability.

Transcription in Prokaryotes

Initiation

Transcription initiation in prokaryotes involves the binding of RNA polymerase to specific DNA sequences called promoters.

• Promoter: DNA sequence upstream of the gene; contains -10 (Pribnow box) and -35 regions recognized by the sigma (σ) factor.

• Sigma Factor: Subunit of RNA polymerase that facilitates promoter recognition and binding.

• Initiation Complex: Formed when RNA polymerase holoenzyme binds to the promoter.

Elongation

During elongation, RNA polymerase synthesizes RNA in the 5' to 3' direction, using the DNA template strand.

• RNA Synthesis: Nucleotides are added to the 3' end of the growing RNA chain.

• Template Strand: The DNA strand used as a template for RNA synthesis.

Termination

Transcription termination in prokaryotes can occur via two main mechanisms:

• Rho-independent termination: Formation of a GC-rich hairpin loop in the RNA followed by a string of uracils causes RNA polymerase to dissociate.

• Rho-dependent termination: The Rho protein binds to the RNA and moves toward the polymerase, causing dissociation when it catches up.

Transcription in Eukaryotes

Key Differences from Prokaryotes

Eukaryotic transcription is more complex, involving multiple RNA polymerases and extensive post-transcriptional modifications.

• RNA Polymerase II: Synthesizes mRNA in eukaryotes.

• Promoters and Enhancers: More complex regulatory sequences.

• Transcription Factors: Proteins required for initiation and regulation.

Post-Transcriptional Modifications

• 5' Capping: Addition of a 7-methylguanosine cap to the 5' end of mRNA for stability and ribosome recognition.

• 3' Polyadenylation (Poly-A Tail): Addition of a poly-A tail to the 3' end for stability and export from the nucleus.

• Splicing: Removal of non-coding introns and joining of exons by the spliceosome.

• Example: The human β-globin gene undergoes splicing to remove two introns from its pre-mRNA.

Translation in Prokaryotes

Initiation

• Shine-Dalgarno Sequence: Ribosome binding site on mRNA, complementary to 16S rRNA, positions the ribosome for translation initiation.

• Initiation Factors (IFs): Proteins (IF1, IF2, IF3) that assist in the assembly of the initiation complex.

• Start Codon: Usually AUG, codes for formylmethionine (fMet) in prokaryotes.

Elongation

• Elongation Factors (EFs): EF-Tu, EF-Ts, and EF-G facilitate tRNA entry, translocation, and peptide bond formation.

• Direction: Polypeptide synthesis proceeds from the N-terminus to the C-terminus.

• Enzymatic Activity: Peptidyl transferase activity of the 23S rRNA (ribozyme) in the large ribosomal subunit catalyzes peptide bond formation.

Termination

• Stop Codons: UAA, UAG, UGA; recognized by release factors (RFs) that promote polypeptide release.

• Ribosome Disassembly: Ribosomal subunits dissociate after translation is complete.

Ribosome Structure and Function

• Prokaryotic Ribosome: 70S, composed of 50S (large) and 30S (small) subunits.

• Function: Coordinates mRNA decoding and peptide synthesis.

Relationship of DNA, Template, Coding, mRNA, and Polypeptide

• Template Strand: DNA strand used for mRNA synthesis.

• Coding Strand: DNA strand with the same sequence as mRNA (except T for U).

• mRNA: Carries the genetic code from DNA to ribosome.

• Polypeptide: Sequence of amino acids determined by mRNA codons.

Aminoacyl tRNA Synthetases

• Function: Enzymes that attach the correct amino acid to its corresponding tRNA ("charging").

• Specificity: Each synthetase is specific for one amino acid and its tRNAs.

Wobble Hypothesis

• Definition: Flexibility in base pairing at the third codon position allows some tRNAs to recognize multiple codons.

• Significance: Reduces the number of tRNAs needed to decode all codons.

• Example: tRNAArg can pair with codons CGU, CGC, CGA, and CGG due to wobble at the third position.

Mutations and DNA Repair

Types of Mutations

• Point Mutation: Change in a single nucleotide (substitution, insertion, or deletion).

• Missense Mutation: Alters one amino acid in the protein.

• Nonsense Mutation: Creates a premature stop codon.

• Silent Mutation: No change in amino acid sequence.

• Frameshift Mutation: Insertion or deletion shifts the reading frame.

• Randomness: Most mutations occur spontaneously and randomly.

Ames Test

• Purpose: Assesses the mutagenic potential of chemical compounds using Salmonella bacteria unable to synthesize histidine.

• Principle: Mutagenic chemicals increase the rate of back-mutation, allowing growth on histidine-free medium.

Transposons

• Definition: DNA sequences that can move (transpose) within the genome.

• Types: Insertion sequences (IS elements), composite transposons.

• Effect: Can cause mutations and genome rearrangements.

DNA Repair Mechanisms

• Direct Repair: Enzymes directly reverse DNA damage (e.g., photolyase repairs thymine dimers).

• Excision Repair: Damaged DNA is removed and replaced (base excision repair, nucleotide excision repair).

• Mismatch Repair: Corrects errors missed by DNA polymerase proofreading.

Gene Regulation in Prokaryotes

Operons

• Definition: A cluster of genes under the control of a single promoter and operator, transcribed as a polycistronic mRNA.

• Components: Promoter, operator, structural genes, regulatory gene.

• Example: The lac operon regulates lactose metabolism in E. coli.

• Regulation: Inducible (lac operon) or repressible (trp operon) systems.

Other Regulatory Elements

• Suppressor: Mutation that counteracts the effect of another mutation.

• Riboswitch: mRNA element that binds small molecules to regulate gene expression.

• sRNA (small RNA): Non-coding RNAs that regulate gene expression post-transcriptionally.

• Attenuator: Sequence that causes premature termination of transcription in response to metabolite levels (e.g., trp operon).

• Antisense RNA: RNA molecule complementary to mRNA, inhibits translation.

• In cis vs. In trans: In cis refers to regulation by elements on the same DNA molecule; in trans refers to regulation by diffusible products (e.g., proteins or RNAs from other loci).