Bacteril transcription (L11)

Question 1

What is the ‘antisense’ strand

Answer 1

the Template strand used in transcription.

Question 1

What bases does RNA use

Answer 1

Adenine, cytosine, guanine and Uracil instead of Thymine

Question 2

Why is Uracil not in DNA

Answer 2

Cytosine undergoes spontaneous deamination, which produces Uracil, but enzyme uracil-DNA glycosylase removes the Uracil in DNA, and is repaired by DNA polymerases.

Question 4

What are the three major classes of bacterial RNA

Answer 4

mRNA, tRNA, rRNA

Question 5

what is mRNA

Answer 5

messenger RNA, codes for proteins

Question 6

What is tRNA?

Answer 6

transfer RNA, used to match codon to its complementary amino acid in protein synthesis.

Question 7

What is rRNA?

Answer 7

ribosomal RNA, constituents of ribosomes, used in protein synthesis.

Question 8

What is the gene being transcripted called?

Answer 8

An operon

Question 9

Name all parts of an operon, downstream.

Answer 9

promoter region, which includes the operator region, protein coding sequences, terminator sequence.

Question 10

What is the role of the promoter region in an operon.

Answer 10

it is the binding site for RNA Polymerase

Question 11

what is the role for the operator region?

Answer 11

binding site for transcription factors. determines whether the gene is expressed depending on conditions. (‘on/off’ switch)

Question 12

What is the function of the terminator sequence.

Answer 12

transcription ends here.

Question 13

what does polycistronic mean?

Answer 13

multiple genes, in this context the operon is polycistronic, as it contains multiple protein coding sequences.

Question 14

What is the STRUCTURAL difference between core RNA Polymerase and the holoenzyme RNA Polymerase

Answer 14

holoenzyme includes a sigma subunit - in addition to alpha, beta, omega subunits

Question 15

What is the difference in function between the core and holoenzyme RNA Polymerase.

Answer 15

holoenzyme RNA Polymerase is essential to bind to the promoter region. core RNA Pol is non specific but allows for transcription as it is not bound to the promoter region.I

Question 16

What was the method to identify the promoter region

Answer 16

addition of small amount of DNase to create ‘ nicks’ in DNA, and in an electropherisis, will display fragments, DNase will not ‘nick’ where the RNA Pol sits, showing a ‘footprint’ of protected sequence i.e the promoter region. There are two protected regions

Question 17

why are there two protected areas during the promoter region test

Answer 17

because the sigma unit in holoenzyme RNA Pol has 2 contact points.

Question 18

Where are the protected sequences on the operon.

Answer 18

-10bp and -35bp

Question 19

What are the 3 stages of transcription

Answer 19

Initiation, elongation, termination

Question 20

What is the inititation stage of transcription

Answer 20

core RNA polymerase binds to DNA non-specifically
sigma unit then binds to the core polymerase, which allows it to bind to the specific promoter region based on WHICH sigma subunit was bound to the core enzyme.

RNA pol pulls downstream dna towards itself, scrunching the DNA (i.e ‘abortive initiation’) until bound to the promotor region and DNA opens at -10bp, promotor sequence is now OPEN.

this causes NEGATIVE supercoiling upstream, POSITIVE supercoiling downstream, which topoisomerases deal with

the sigma subunit detatches after about 10 or 15 nucleotides have been synthesised.

Question 21

What is the elongation stage?

Answer 21

the synthesis of the new RNA .

Question 22

What is the termination stage?

Answer 22

there are two mechanism:
p-independent and p dependant
independant- terminatior sequence in the RNA is recognised. common sequence is palindromic GC rich sequence followed by T rich sequence, which when transcripted will form a hair pin, causing the RNA Pol to dissociate.
dependent- p protein is required to break the RNA:DNA duplex in the transcription bubble. protein binds to the RNA strand and chases RNA pol and disrupts it causing dissociation.