Lecture 15

Question 1

What number is the start site of transcription usually given?

Answer 1

+1

Question 2

What are the three stages of transcription?

Answer 2

Initiation, Elongation, & Termination

Question 3

What is transcription initiation?

Answer 3

A collection of proteins called transcription factors mediate the binding of RNA polymerase and the initiation of transcription. Only after certain transcription factors are attached to the promoter does the RNA polymerase bind to it. The completed assembly of transcription factors and RNA polymerase bind to the promoter, forming a transcription initiation complex.

Question 4

What is transcription elongation?

Answer 4

One strand of the DNA, the template strand (or noncoding strand), is used as a template for RNA synthesis. As transcription proceeds, RNA polymerase traverses the template strand and uses base pairing complementarity with the DNA template to create an RNA copy. Although RNA polymerase traverses the template strand from 3’ → 5’, the coding (non-template) strand and newly formed RNA can also be used as reference points, so transcription can be described as occurring 5’ → 3’. This produces an RNA molecule from 5’ → 3’, an exact copy of the coding strand (except that thymines are replaced with uracils, and the nucleotides are composed of a ribose (5-carbon) sugar where DNA has deoxyribose (one fewer oxygen atom) in its sugar-phosphate backbone).

Question 5

What is transcription termination?

Answer 5

Transcription termination in eukaryotes is less understood but involves cleavage of the new transcript followed by template-independent addition of As at its new 3’ end, in a process called polyadenylation

Question 6

What is polyadenylation?

Answer 6

Polyadenylation is the addition of a poly(A) tail to a primary transcript RNA. The poly(A) tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA (mRNA) for translation. The 3’-most segment of the newly made pre-mRNA is first cleaved off by a set of proteins; these proteins then synthesize the poly(A) tail at the RNA’s 3’ end. In some genes, these proteins may add a poly(A) tail at any one of several possible sites. Therefore, polyadenylation can produce more than one transcript from a single gene (alternative polyadenylation)

Question 7

Why is Rifampin used in treating tuberculosis?

Answer 7

Rifampin inhibits bacterial RNA polymerase by binding to it and altering its conformation, but Rifampin does not bind to human RNA polymerase

Question 8

What are the two transcription initiatory regions in prokaryotes?

Answer 8

Pribnow box (TATAAT) and the -35 sequence (TTGACA)

Question 9

What mediates gene expression in transcription?

Answer 9

The interaction between repressor and inducer molecules (such as the lac operon repressor + lactose)

Question 10

What is the function of the lac operon?

Answer 10

It is used to breakdown lactose into sugars for cellular metabolism

Question 11

What part of the lac operon encodes permease?

Answer 11

Lac Y gene

Question 12

What are the two major proteins produced by the lac operon and what are their functions?

Answer 12

Permease - Acts as a transport protein to allow lactose to enter the cell at a greater rate
B-galactoside - Breaks down lactose within the cell

Question 13

What part of the lac operon encodes the lac repressor?

Answer 13

Lac I gene

Question 14

What part of the lac operon encodes B-galactosidase?

Answer 14

Lac Z gene

Question 15

What part of the lac operon encodes B-galactoside transacetylase?

Answer 15

Lac A gene

Question 16

What does B-galactoside transacetylase do?

Answer 16

β-galactoside transacetylase is an enzyme that transfers an acetyl group from acetyl-CoA to β-galactosides. Coded by the gene (lacA) in the lac operon of Escherichia coli. It is thought as a way to excrete unmetabolizable sugars

Question 17

What are two byproducts of cleaving lactose?

Answer 17

Galactose and glucose

Question 18

What are two requirements to activate the lac operon?

Answer 18

The presence of lactose and the absence of glucose

Question 19

What is the function of DNA footprinting?

Answer 19

To assess where on DNA a particular binding protein will bind

Question 20

What is the catabolite activator protein (CAP) associated with the lac operon do?

Answer 20

In the presence of glucose, the catabolite activator protein (CAP), required for production of the enzymes, remains inactive, and EIIAGlc shuts down lactose permease to prevent transport of lactose into the cell.

Question 21

What does the term diauxie mean?

Answer 21

Rather than metabolizing the two available sugars simultaneously, microbial cells commonly consume them in a sequential pattern, resulting in two separate growth phases.

Question 22

Why does RNA polymerase have weak affinity for the lac operon in general?

Answer 22

The lac operon promoters differ from promoter consensus sequences causing the affinity of RNA polymerase to be weaker. Therefore, the catabolite activator protein (CAP) is required to promote activation of the lac operon

Question 23

How does a lack of glucose recruit catabolite activator protein (CAP)?

Answer 23

When glucose becomes depleted, it makes adenylate cyclase, which turns ATP into cAMP. When cAMP levels rise in the cell, it binds to transcription factor CAP and activates transcription

Question 24

What does the trp operon do?

Answer 24

The trp operon makes tryptophan

Question 25

Normally, is the trp operon activated or deactivated?

Answer 25

Activated

Question 26

What causes the trp operon to be deactivated?

Answer 26

In the presence of excess tryptophan (co-repressor), trp binds to the trp repressor causing it to be activated and bind to the trp operon. This prevents more trp from being produced

Question 27

What is auto-regulation and how does it relate to the trp operon?

Answer 27

The amount of trp regulates trp production in the cell. Therefore, tryptophan is auto-regulating itself.

Question 28

Why does a hairpin formation between the 3 and 4 sequence in the trp L sequence cause termination while a hairpin formation between the 2 and 3 sequence in the trp L sequence allows for continuation of transcription/translation?

Answer 28

A hairpin formed between the 3 and 4 sequence in the trp L sequence has a tail of U’s which result in a complementary poly A tail. poly A tails indicate the presence of mature mRNA and terminate transcription

Question 29

In eukaryotes, what is the function of RNA Polymerase 1?

Answer 29

Produces 3 out of 4 rRNAs

Question 30

In eukaryotes, what is the function of RNA Polymerase 2?

Answer 30

Produces all mRNA

Question 31

In eukaryotes, what is the function of RNA Polymerase 3?

Answer 31

Produces all tRNA and 1 out of 4 rRNAs

Question 32

What does the mushroom Amanita phalloides inhibit?

Answer 32

RNA Polymerase 2, thereby inhibiting the production of mRNA