Transcription & RNA Processing

Question 1

Define gene expression.

Answer 1

The transfer of information from DNA (where it’s stored) to RNA.

Question 2

How is transcription different in eukaryotes and prokaryotes?

Answer 2

In prokaryotes, RNA is transcribed directly from the genome onto the ribosomes. In eukaryotes the process of transcribing RNA is much more complex and originates in the nucleus., starting with the primary transcript, then the mRNA.

Question 3

What are the five stages of the transcription cycle?

Answer 3

1. Template binding
2. Chain initiation
3. Promoter clearance
4. Chain elongation
5. Chain termination and RNAP release

Question 4

How does the presence of lactose affect bacterial cells’ gene expression?

Answer 4

The presence of lactose in the medium induces the synthesis of the enzyme B-galactosidase. This is an example of how bacterial cells selectively express genes to use the available resources effectively.

Question 5

Define bacterial operons.

Answer 5

An operon is a functional complex of genes containing the information for enzymes of a metabolic pathway. Operons allow a set of genes with similar functions to all be turned on or off at the same time–kind of like a master light switch.

Question 6

What do the structural genes of an operon code for?

Answer 6

They code for enzymes and are translated from a single mRNA that is usually polycistronic (encodes for more than one protein).

Question 7

Where does the RNA polymerase bind on an operon?

Answer 7

The promoter.

Question 8

What is the operator of an operon?

Answer 8

The site next to the promoter where the regulatory protein can bind.

Question 9

What is the repressor of a bacterial operon?

Answer 9

This repressor binds to a specific DNA sequence (the operator) to determine whether or not a particular gene is transcribed. RNA polymerase is unable to bind to a promoter if the repressor is bound.

Question 10

In the tryptophan biosynthetic operon (which is an example of a repressible operon), when is the repressor protein active and able to bind to the operator?

Answer 10

The repressor protein is only active (and able to bind to the operator) when there is excess tryptophan co-repressor. When Trp is not in excess, the repressor is inactivated and synthesis is derepressed.

Question 11

In the lactose catabolic operon (which is an example of an inducible operon) what activates the operon?

Answer 11

The inducible operon is turned on in the presence of lactose (inducer) which needs to be digested. Lactose binds to the repressor, changing its conformation and making it unable to bind to the operator. The repressor can bind to the operator and prevent transcription in the absence of lactose.

Question 12

What does catabolite repression allow the cell to do?

Answer 12

It allows the cell to use preferred energy sources (usually glucose).

Question 13

How does glucose affect cAMP levels?

Answer 13

When glucose falls, cAMP rises.

Question 14

How does cAMP affect RNA binding to the promoter?

Answer 14

cAMP acts by binding to a cAMP receptor protein (CRP)

Binding of CRP-cAMP to the lac control region changes the conformation of DNA, allowing DNA polymerase to transcribe the lac operon when the repressor is no longer an operator. Without cAMP, RNA binding to the promoter is very poor/weak.

Question 15

What two conditions must be met to use the lactose operon?

Answer 15

1. The presence of lactose with the ability to bind to the lactose oppressor to remove its ability to bind to the operator.
2. An adequate promoter for RNA polymerase, which only occurs when cAMP levels are high and able to bind to CRP, which binds to a site upstream from the promoter.

Question 16

What is the function of RNA polymerase (RNAP)?

Answer 16

RNAP is responsible for incorporating ribonucleoside triphosphates into RNA strands whose sequence is complementary to the DNA template.

Question 17

What direction does RNAP move?

Answer 17

RNAP moves in a 3’ –> 5’ direction.

Question 18

What direction does the nascent (newly synthesized) RNA grow in?

Answer 18

5’ –> 3’.

Question 19

How are the template strand and the coding strand related?

Answer 19

They are complementary and antiparallel. The template strand has the opposite orientation to the coding strand and the nascent RNA chain.

Question 20

What is the function of the five subunits of bacterial RNAP?

Answer 20

Bacterial RNA polymerase has five subunits that can bind randomly to transcribe RNA. Association with the sigma factor forms a complete haloenzyme with these five core subunits to allow promoter binding and strand separation → transcription initiation.

Question 21

What are the two basic elements of a bacterial promoter?

Answer 21

1. The 35 bp upstream element.
2. The 10 bp (Pribnow Box) upstream element.

Question 22

How is bacterial transcription terminated when it is rho-dependent?

Answer 22

Rho purses the polymerase, the hairpin forms, the polymerase pauses, the rho catches up, and the rho causes termination.

Question 23

What drives RNA formation of hairpin loops?

Answer 23

Bacterial transcription termination sequences.

Question 24

Why is eukaryotic transcription more complex than prokaryotic transcription?

Answer 24

It is tissue/temporally specific. This means that depending on the type of cell of the organism (for example, neuron vs liver cell), different genes must be transcribed. Time and location tell the cells which genes to activate and which to repress.

Question 25

What does the transcription factor TBP do?

Answer 25

It recognizes the TATA box and binds to that sequence during the formation of pre-initiation complexes.

Question 26

What do TBP and TAFs accomplish when bound together?

Answer 26

Together, they provide a binding site for RNA polymerase II binding.

Question 27

What is TFIIH?

Answer 27

The critical component that will not come in to the pre-initiation complex unless all of the other TFs are bound in their place.

Question 28

What step is required for the activation of transcription and the transition from inactive pre-initiation complex to an active initiation complex?

Answer 28

The carboxy terminal tail of polymerase II has a series of seven amino acids (heptapeptide) repeated. At the site of the 5th amino acid is a serine, and it is the site at which the kinase of TFIIH phosphorylates; this is the important step.

Question 29

What is the limiting step in the formation of the basal transcription initiation complex?

Answer 29

TFIIB recruitment.

Question 30

What raises transcription from basal to activated levels?

Answer 30

TBP associated factors (TAFs) stabilize the TBP complex by linking to upstream enhancer binder proteins.

Question 31

Define transcription activators.

Answer 31

Transcription activators/factors are multi-functional proteins with specific domains that can act independently.

Question 32

What end of mRNAs is usually capped, and what is it capped with? What is on the other end?

Answer 32

The 5’ end of mRNAs is usually capped with 7-methyl-guanosine. The other end has a 3’ poly-A tail.

Question 33

What are the three purposes of the mRNA cap?

Answer 33

1. Stability for the mRNA
2. Aids in transport to the cytoplasm
3. Helps to initiate translation.

Question 34

What do capping enzymes do?

Answer 34

They take hnRNAs (heteronuclear RNAs) and transition them to mRNAs.

Question 35

In the processing of mRNA caps, what does triphosphates do?

Answer 35

It cleaves the terminal triphosphate to a diphosphate, creating a substrate for a group of enzymes known as guanyltransferases.

Question 36

In the processing of mRNA caps, what do guanyltransferases do?

Answer 36

They add GMP in an inverted orientation to create a 5’-5’ triphosphate bond.

Question 37

In the processing of mRNA caps, what do RNA methyltransferases do?

Answer 37

They methylate the inverted guanosine at N7 and the internal nucleoside.

Question 38

What two modifications occur prior to the completion of splicing on the 3’ end of mRNAs?

Answer 38

1. Cleavage to yield a unique 3’ OH.
2. Addition of about 200 adenylate molecules to generate poly A+ tail.

Question 39

What is mRNA export?

Answer 39

The process by which mature mRNAs leave the nucleus for cytoplasmic translation.

Question 40

How does the view of a nuclear pore differ between the nuclear and cytoplasmic faces?

Answer 40

From the cytoplasmic face, these pores have a peripheral ring structure. From the nuclear face, they have an inner ring basket structure.

Question 41

What is the nuclear pore complex?

Answer 41

This complex is made up of 8 copies of about 30 highly conserved proteins known as nucleoporins. It mediates nuclear export and nuclear import.