Lecture 7 Transcription and Gene Regulation

Question 1

What is a gene?

Answer 1

The sum of all transcribed and non-transcribed regions that are necessary to properly express a gene (must consider not just the coding region, but also the regulatory region etc)

Question 2

The transcribed region of a gene is referred to as the:

Answer 2

Transcription unit

Question 3

What is the open reading frame (ORF) of the transcription unit?

Answer 3

The coding region (operon-derived mRNAs harbor more than one ORF)

Question 4

Some genes encode _____ that are no translated

Answer 4

RNAs

Question 5

Not everything that is transcrbed is necessarily coding: provide an examp

Answer 5

5’ and 3’ UTR (untranslated regions) and introns

Question 6

Regulatory sequences are commonly located in the _________ regions of a gene but sequences with important ______ functions may reside in the transcription unit and coding region

Answer 6

Regulatory sequences are commonly located in theNON-TRANSCRIBED regions of a gene but sequences with important REGULATORY functions may reside in the transcription unit and coding region

Question 7

Transcription occurs via _____ _______

Answer 7

RNA polymerase

Question 8

During transcription _____ the DNA is melted; Transcription then occurs in a “______”

Answer 8

During transcription INITIATION the DNA is melted; Transcription then occurs in a “TRANSCRIPTION BUBBLE”

Question 9

What is the nontemplate strand?

Answer 9

Coding strand = “sense” strand Identical in sequence to the mRNA 5’-3’

Question 10

What is the template strand?

Answer 10

Non-coding strand = “antisense” strand 3’-5’

Question 11

Principle difference between bacterial and eukaryotic gene regulation?

Answer 11

• The ground state is ON in bacterial gene regulation (DNA is accessible at all times)
  
  • Gene requires repression to turn “off”
• Whereas ground state in eukaryotes is OFF (because of nucleosomes = DNA is inaccessible)
  
  • Gene requires activation “on”

Question 12

Bacterial RNA Polymerase is a multi-subunit protein complex

• ____different gene products required for the ____ enzyme
• Two ___ subunits which are identical and encoded by the same gene
• ___, ____ and _____ are encoded by three additional genes
• _____ _____ is needed for specific binding of the RNA polymerase to the promotor

Answer 12

Bacterial RNA Polymerase is a multi-subunit protein complex

• Four different gene products required for the Core enzyme
• Two Alpha subunits which are identical and encoded by the same gene
• omega, beta and beta prime are encoded by three additional genes
• sigma factor is needed for specific binding of the RNA polymerase to the promotor

Question 13

What is Holoenzyme?

Answer 13

Core enzyme (of RNA polymerase) plus Sigma factor

Question 14

Sigma factor has the ability to bind to the ____ ____ _____ which drastically improves the ability to bind to promotors

Answer 14

Sigma factor has the ability to bind to the Core RNA Polymerase which drastically improves the ability to bind to promotors

Question 15

What is a promoter?

Answer 15

Region of a gene containing regulatory sequences that are close to (or contain) the transcription start site

Required for transcription initiation via RNA polymerase

Question 16

What is positive regulation?

Answer 16

Involves an activator

• activator bind to activator binding site
  
  • allows RNA polymerase to bind to promotor

Question 17

What is negative regulation?

Answer 17

Involves a Repressor

• Bind to operator sequences (part of promoter)
• Either prevents binding of RNA polymerase OR blocks progress of RNA polymerase

Question 18

What is the Operator Sequence?

Answer 18

• part of promoter
• designed to interfere with transcription (ie turns gene off)

Question 19

What is a ligand? What is it’s role in transcription?

Answer 19

Small units (metabolites/hormones/peptide hormones) that can bind to activators (via ligand-binding site/allosteric site) = change conformation of protein = Activating: Promote binding of DNA or Inhibiting: Promote release of DNA from activator-binding site

Question 20

What is an allosteric site?

Answer 20

Region of protein to which a small molecule can bind (the ligand) resulting in a conformational change of the protein that modifies the activity of the protein

The result can be inhibitory or activating

Question 21

The lac operon comprises three genes:

Answer 21

Beta-galactosidase

Permease

Transacetylase

Question 22

What are operons?

Answer 22

A group of genes that are adjacent to each other and transcribed together (under control of a single regulatory region)

Question 23

What result on mRNA does an operon have?

Answer 23

Resulting mRNA is polycistronic

Question 24

Are operons found in eukaryotes or bacteria?

Answer 24

bacteria

Very rare in eukaryotes

Question 25

An mRNA that can encode more than one polypeptide separately within the same RNA molecule is called:

Answer 25

Polycistronic

Question 26

What are operators?

Are they found in bacteria or eukarya?

Answer 26

Region of DNA that acts as a binding site for a repressor protein

• Found in bacteria (has not been identified in eukaryotes)

Question 27

What are the three structural genes of the Lac operon? What do they do?

Answer 27

1. lacZ: beta-galactosidase
   
   • cleaves lactose into galactose and glucose
   • also produces beta1,6-allolactose
2. lacY: transports lactose into the cell
3. lacA: transfers acetyl group to certain sugars

Question 28

What are the two regulatory genes of the Lac operon?

Answer 28

1. lacI: lac repressor - not part of the lac operon but the lacI gene lies nearby (upstream of lac operon)
2. CAP: catabolite activator protein
   
   • also known as CRP (cAMP receptor protein)

Question 29

What are the two Substrates/Effectors (ligands/inducers) of the lac operon?

Answer 29

1. beta-1,6-allolactose: a transient byproduct of beta-galactosidase activity
   
   • Triggers conformational change in lac repressor
2. cAMP: cyclic adenosine monophosphate
   
   • Allosteric effector for CAP/CRP - made when glucose is low

Question 30

What two conditions must be met to have the lac operon induced?

Answer 30

1. Lactose is present in the growth medium
2. Glucose is absent from the growth medium

Question 31

If glucose is present in the growth medium what happens?

Answer 31

Glucose is preferred sugar: Basal transcription

Question 32

What is Basal transcription?

Answer 32

• occurs when RNA polymerase binds to DNA in the absence of activators and repressors (RNA polymerase Is neither specifically recruited to nor prevented from binding to the promotor)

Question 33

What is the general mechanism of operator function?

Answer 33

• competition for promotor occupancy
• operator-bound repressor blocks binding or transcription of RNA polymerase
• Operator doesn’t have to be located between -10 and -35 elements
• Basal level of transcription generally refers to random binding of RNA Pol to the promoter in the absence of activators and repressors

Question 34

Under what conditions of the lac operon might you see basal transcription?

Answer 34

Basal transcription can occur in a repressed state

Question 35

Lac operon overview:

If we have neither the CAP(activator) nor Lac repressor what do we see?

Can happen in ___glucose, ____lactose conditions

Answer 35

Basal transcription - RNA polymerase will bind occasionally

Can happen in _+_glucose, _+_lactose conditions

Question 36

In +glucose, -Lactose conditions what would we see?

Answer 36

No lactose in the medium = attracts binding of Lac repressor = operon is off

Question 37

What would happen in -glucose, -lactose conditions?

Answer 37

No lactose = attracts Lac repressor = Operon off

Question 38

What would we observe in -Glucose, +lactose conditions?

Answer 38

Operon ON = this is the only state where we see a fully activated operon

Presence of lactose in the absence of glucose = CAP binds and repressor is removed