REGULATION OF GENE EXPRESSION IN PROKARYOTES Flashcards
LEARNING OBJECTIVES
- Discuss the concept of central dogma
- provide a general overview of transcription and RNA polymerase structures
- explain functional elements in bacterial promoters and how they affect interaction with RNA polymerase and protein abundance
- introduce concept of gene reporter and compare 2 types of reporters
central dogma
Genetic information flows from DNA → RNA (transcription) → Protein (translation).
Reverse transcription: RNA → DNA (no protein → RNA or protein → DNA).
In prokaryotes, transcription and translation are coupled (occur simultaneously).
transcription in eukaryotes
RNA polymerase synthesizes RNA from a DNA template.
Requires four ribonucleotides: ATP, GTP, CTP, UTP.
Genes are transcribed into pre-mRNA, introns are spliced out to form mRNA.
mRNA leaves the nucleus for translation at the ribosome.
gene structure in eukaryotes
Promoter: Upstream of the coding sequence, contains regulatory elements like the TATA box.
Coding sequence: Contains the instructions for protein synthesis.
Terminator: Downstream of the coding sequence; signals end of transcription.
Translation begins at AUG (start codon) and stops at one of the stop codons (TAA, TAG, TGA).
operons in prokaryotes
Operon: A group of genes regulated by a single promoter.
Produces polycistronic mRNA (multiple proteins) or monocistronic mRNA (one protein).
Genes in an operon are transcribed together, and proteins are often functionally related.
stages of transcription in prokaryotes
Initiation: Sigma factor recognizes promoter (-35 and -10 sequences) and initiates transcription.
Elongation: RNA polymerase synthesizes RNA in the 5’ to 3’ direction.
Termination: Transcription ends at the terminator, RNA polymerase detaches.
RNA polymerase
Prokaryotes: Only one RNA polymerase for all genes.
Eukaryotes: Three RNA polymerases (RNA Pol I, II, III), each for different types of RNA.
promoter strength
Strong promoters: Frequently occupied by RNA polymerase, more transcripts, more protein.
Weak promoters: Less frequent binding, fewer proteins.
Sequence variations in -35 and -10 regions affect promoter strength.
regulating gene expression
Transcriptional Level:
Post-transcriptional:
Translational:
Post-translational:
negative gene regulation
Repressors bind to the operator region, preventing transcription.
Default: ON, repressor turns transcription OFF.
positive gene regulation
Activators help RNA polymerase bind to weak promoters, allowing transcription.
Default: OFF, activator turns transcription ON.
lac operon example
Encodes proteins for lactose metabolism.
Negative regulation: LacI repressor binds to operator when glucose is present, stopping transcription.
Positive regulation: When glucose is low, CAP (with cAMP) binds to promoter, facilitating transcription if lactose is present.
allosteric regulation
Effector molecules (e.g., alloLactose, cAMP) bind to proteins and change their shape, affecting function.
LacI and CAP are controlled by allosteric transitions (inhibition or activation).
Lac 1 changes to non conformational when aLAC binds - allosteric inhibition
CAP - Changes to conformational when cAMP binds - allosteric activation
lac operon key points on regulation
Negative Regulation: No lactose = No transcription (LacI repressor binds)
Positive Regulation: Low glucose = CAP-cAMP helps RNA polymerase transcribe when lactose is available.
identifying the strength of promoter using reporter genes
approach 1 - reporter gene encodes fluorescent protein. the stronger the promoter, the more GFP mRNA, the more GFP protein, brighter GFP signal
approach2 - reporter gene encodes an enzyme with easy to measure activity - beta-galactosidase . stronger promoter, more lacZ mRNA, more b-gal enzyme, more reaction to product.
