REGULATION OF GENE EXPRESSION IN PROKARYOTES

Question 1

LEARNING OBJECTIVES

Answer 1

1. Discuss the concept of central dogma
2. provide a general overview of transcription and RNA polymerase structures
3. explain functional elements in bacterial promoters and how they affect interaction with RNA polymerase and protein abundance
4. introduce concept of gene reporter and compare 2 types of reporters

Question 2

central dogma

Answer 2

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).

Question 3

transcription in eukaryotes

Answer 3

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.

Question 4

gene structure in eukaryotes

Answer 4

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).

Question 5

operons in prokaryotes

Answer 5

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.

Question 6

stages of transcription in prokaryotes

Answer 6

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.

Question 7

RNA polymerase

Answer 7

Prokaryotes: Only one RNA polymerase for all genes.

Eukaryotes: Three RNA polymerases (RNA Pol I, II, III), each for different types of RNA.

Question 8

promoter strength

Answer 8

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.

Question 9

regulating gene expression

Answer 9

Transcriptional Level: More RNA = More protein. express genes they need and activate others in response to signals, using alternative sigma factors

Post-transcriptional: RNA stability affects protein levels.

Translational: Premature termination leads to less protein.

Post-translational: Protein modifications (e.g., phosphorylation, ubiquitination).

Question 10

negative gene regulation

Answer 10

Repressors bind to the operator region, preventing transcription.

Default: ON, repressor turns transcription OFF.

Question 11

positive gene regulation

Answer 11

Activators help RNA polymerase bind to weak promoters, allowing transcription.

Default: OFF, activator turns transcription ON.

Question 12

lac operon example

Answer 12

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.

Question 13

allosteric regulation

Answer 13

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

Question 14

lac operon key points on regulation

Answer 14

Negative Regulation: No lactose = No transcription (LacI repressor binds)

Positive Regulation: Low glucose = CAP-cAMP helps RNA polymerase transcribe when lactose is available.

Question 15

identifying the strength of promoter using reporter genes

Answer 15

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.

Question 16

explanation of lac operon regulation

Answer 16

When glucose available, the Lac1 repressor binds to operator to prevent RNA polymerase from binding to the Lac Operon. Also glucose prescence inhibts the ATP –>cAMP.
- However when lactose present, lactose is converted into alloLACTOSE which binds to the Lac1, causing it to detach from the operator, allowing RNA polymerase to conduct transcription.
But for transcription to work well, CAP (catabolite activator protein) is needed as an activator to anchor RNA polymerase to the promoter.
- CAP cannot bind to the DNA without cyclic AMP, which is only made from ATP–> cAMP when glucose levels are low as glucose inhibits the product of cAMP.
Thus is glucose levels are low, cAMP binds to CAP, allowing it to bind to the activator binding site to anchor RNA polymerase and transcription can occur.

The lac operon promoter is very weak