Lecture 20

Question 1

Transcription

Answer 1

Transfers info from DNA to RNA. 5’-3’. There is a template strand, first nucleotide where 3’ OH group ‘attacks’ innermost phosphoryl group of the ribonucleotide creating a phosphodiester bond. Driven forward by hydrolysis of pyrophosphate groups to inorganic phosphate. Same as DNA synthesis. What’s different is the enzymes. RNA polymerase involved and can occur without primers.

Question 2

mRNA

Answer 2

Encodes proteins

Question 3

tRNA

Answer 3

Carries amino acids

Question 4

rRNA

Answer 4

Catalytic component of ribosomes

Question 5

RNA production

Answer 5

Make by RNA polymerase in prokaryotes.

Question 6

Requirements for DNA template

Answer 6

What RNA polymerase copies. AKA antisense strand. Is complementary to RNA transcript. Coding (sense) strand is same as RNA except T instead of U.

Question 7

Requirements for ribonucleoside triphosphates

Answer 7

ATP, GTP, UTP, CTP

Question 8

Requirements for RNA polymerase

Answer 8

In bacteria, fairly large where 4 core subunits and 1 transient subunits. Cofactors incl. Mg2+

Question 9

Promoter

Answer 9

DNA sequences that directs RNA polymerase to the proper site to begin.

Question 10

Initiation

Answer 10

Transcription is initiated at promoter site. In bacteria, there are 2 common 6bp long sequences known as the -10 (TATAAT) and -35 (TTGACA) elements which are centred at 10 and 35 nucleotides upstream of the start site respectively. These sequences are consensus/average sequences deduced from analysing many promoter sequences. Promoter sequences impact the frequency of transcription. Strong promoter sequences also important. Optimal distance is 17 nucleotides as RNA polymerase fits it nicely.

Question 11

Bacterial promoter sequences

Answer 11

Promoters are recognised by RNA polymerase sigma subunit which lowers affinity of RNA polymerase for general sections of DNA. It allows RNA polymerase to quickly find and dock to promoter region. Once transcription is initiated, sigma is released where it can participate in another round of transcription. Sigma release signals end of initiation. Sigma is released to increase grip on DNA sequence.

Question 12

Elongation

Answer 12

RNA polymerase unwinds DNA and it occurs de novo (no primers) 5’-3’. The first base is either A or G due to stability.

Question 12

Elongation steps

Answer 12

1. RNA polymerase unwinds DNA via helicase in transcription bubbles.
2. RNA strand formed, but, it forms duplex with DNA (~8bp long). SLOW
3. RNA polymerase has limited proof-reading capability. RNA synthesis is more error prone than DNA synthesis.

Question 12

Intrinsic termination

Answer 12

Transcription stops due to signals within the newly synthesised RNA incl. hairpin loop formed palindromic. GC region + 4 uracils as it causes RNA polymerase to pause dissociating mRNA product from the template allowing it to anneal with its OG binding partner (other DNA strand). Transcription bubble closes, dissociating RNA polymerase. C

Question 13

Roh-dependent termination

Answer 13

C-rich section on RNA strand recruits Roh protein which binds to it. It slides across RNA until transcription bubble reached and RNA polymerase bumped. Causes RNA to dissociate from template and DNA anneal.

Question 14

lac-operon

Answer 14

Promoter and operator regulate the expression of these genes. Repressor gene located upstream. When enzymes needed, they are all transcribed at the same time. lac-mRNA would code for all 3 enzymes (called polycistronic mRNA or polygenic mRNA). Each gene has it’s own start and stop codon so they are translated independently.

Question 14

lactose operon

Answer 14

Bacteria use glucose as main energy source but, use lactose when glucose decreases. In order to use lactose, it needs to produce 3 enzymes for the break down and transport of lactose. They are functionally related therefore grouped to increase efficiency of transcription and translation.

Question 15

operon

Answer 15

Grouping of genes that are functionally related under the control of a single promoter.

Question 16

When lactose is absent

Answer 16

When absent in environment, genes are not expressed. The repressor gene is transcribed and translated which is almost always occurring due to increase promoter strength. Repressor protein has high affinity for operator site on lac operon, blocking it’s transcription.

Question 17

Additional regulation by glucose

Answer 17

Lac operon promoter is weak therefore rate of transcription very slow. This is because in the presence of glucose, lactose is the least preferred energy source. Lac expression is increased when [glucose] decreases. Reduction of [glucose] = increase cAMP = cAMP binding to CAP = cAMP/CAP complex binding to CAP-binding site upstream of transcription start site = stimulates RNA poly. recruitment (50x more than promoter region can recruit) = increase transcription

Question 18

When lactose is present

Answer 18

Cue for cell to transcribe enzymes. As B- galactoside always present (small quantities) it breaks lactose down forming allolactose as a byproduct. Allolactose is inducer where it binds and changes affinity of repressor protein of operator site. This shape changes means it cannot block enzyme expression.

Question 19

trip operon

Answer 19

Bacteria can uptake tryptophan from the environment but, can also make it using 5 genes within the trp operon. If needed, all 5 are coded into the same mRNA with their own start/stop codon to allow for independent translation.

Question 20

Presence of tryptophan

Answer 20

trp genes switched off. Tryptophan acts as co-repressor where it binds to repressor and both bind to the operator site to prevent transcription (both must be present).

Question 21

Absence of tryptophan

Answer 21

No tryptophan = no co-repressor = inactive repressor = trp gene transcription

Question 22

antibiotics as transcription inhibitors

Answer 22

Some antibiotics act by inhibiting bacterial transcription e.g Rifampicin blocks channel bacterial RNA polymerase into which the RNA-DNA hybrid must pass and so stops elongation.