Wright Lecture 6: Prokaryotic Gene Regulation

Question 1

Transcription/translation in prokaryotic cells vs. eukaryotic cells

Answer 1

Transcription and translation is coupled in prokaryotic cells but separated in eukaryotic cells

Question 2

Pivotal point in controlling gene expression in prokaryotic cells

Answer 2

Site of transcriptional initiation: promoter

Question 3

Methods to identify the sequence of a bacterial promoter (3)

Answer 3

1. Sequence DNA fragments protected by RNA polymerase from digestion by DNase I
2. Align sequences upstream of ignition site of transcription (comparing between operons)
3. Analyze mutations with different rates of transcription

Question 4

Protecting DNA sequences with RNA polymerase

Answer 4

1. Add RNA polymerase and DNA fragment to test tube allowing protein DNA binding
2. Add DNase to test tube to digest unprotected DNA
3. Purify protected DNA, clone and sequence

Question 5

Mutant consensus sequences

Answer 5

Mutations that change the nucleotide sequence from promoter diminish rate of transcription

Question 6

Three stages of RNA synthesis

Answer 6

1. Initiation
2. Elongation
3. Termination

Question 7

Initiation of RNA synthesis

Answer 7

RNA polymerase recognizes promoter, melts the TATA box and initiates incorporation of first ribonucleoside triphosphate into the nascent RNA

Question 8

Elongation of RNA synthesis

Answer 8

Growth of RNA polymer in 5’ to 3’ direction

Question 9

Termination of RNA synthesis

Answer 9

Synthesis of RNA polymer stops at precisely defined site coded in DNA template

Question 10

RNA polymerase

Answer 10

Multi-subunit enzyme: 2alpha, 1beta, 1beta’, 1omega
Holoenzyme is required for promoter recognition and initiation of transcription
Core enzyme proceeds to complete elongation and termination of RNA transcript

Question 11

Omega (RNA polymerase)

Answer 11

Not essential for transcription but helps stabilize the RNA polymerase

Question 12

Sigma factor (RNA polymerase)

Answer 12

Controls initial binding of RNA polymerase to the promoter

Released from holoenzyme after initiation to participate in recognition of other promoters

Question 13

Holoenzyme (RNA polymerase)

Answer 13

2alpha, 1beta, 1beta’, sigma factor

Required for promoter recognition and initiation of transcription

Question 14

Core enzyme (RNA polymerase)

Answer 14

Holoenzyme release sigma factor after initiation to leave core enzyme: 2alpha, 1beta, 1beta’

Question 15

Integration of RNA polymerase at promoter before initiation

Answer 15

1. Loose binding of holoenzyme
2. RNA polymerase scans double helix
3. Polymerase binds tightly to promoter region
4. Polymerase unwinds DNA
5. Sigma is released
6. Transcription begins

Question 16

Closer-promoter to open promoter

Answer 16

Irreversible step in initiation of transcription

1. Unwinding of promoter and beginning to RNA transcription moving from promoter
2. Sigma factor is released
3. RNA elongation

Question 17

Catabolic pathways

Answer 17

Break down of chemicals

Question 18

Anabolic pathways

Answer 18

Build up of chemicals to end-product

Question 19

Polygenic mRNA

Answer 19

From prokaryotes
Multiple proteins on same operon
Direct translation into polypeptide

Question 20

Tryptophan operon

Answer 20

In prokaryotes: clustered together under control of single promoter
In eukaryote: dispersed throughout the genome, each gene is controlled by a separate promoter

Question 21

Metabolism of lactose

Answer 21

Break down into glucose and galactose by beta-galactosidase

Question 22

Induction of lactose operon

Answer 22

Low levels of beta-galactosidase in cells causes build up of allolactase (lactose isomer) which induces lactose operons

Question 23

Lactose repressor protein

Answer 23

Binds to operator, blocks transcription of operon
Binding of allolactose (reversible) to binding domain causes conformational change that masks DNA binding domain
Induction of lactose operon by lactose

Question 24

Regulatory proteins

Answer 24

In both prokaryotic and eukaryotic gene regulation
Undergo conformational change upon binding small molecule (ligand) that decreases or increases affinity for DNA
Allosteric proteins

Question 25

Allosteric protein

Answer 25

Proteins that undergo a conformational change upon binding a ligand