Lecture 14

Question 1

What does it mean that in bacteria, transcription and translation are coupled?

Answer 1

the process of synthesizing a mRNA molecule (transcription) happens at the same time as the process of using that mRNA to build a protein (translation)

Question 2

What is the main process of transcription?

Answer 2

RNA polymerase uses the DNA template to synthesize mRNA

Question 3

What is the main process of translation?

Answer 3

ribosomes read the mRNA sequence and build a growing polypeptide chain (protein)

Question 4

What does RNA polymerase do in transcription?

Answer 4

Synthesizes RNA from DNA template by binding to a promoter, unwinding the double helix, and joins nucleoside triphosphates (NTPs) by phosphodiester linkages from 5’ to 3’ using DNA as a template

Question 5

What’s the difference between RNA and DNA polymerase?

Answer 5

DNA polymerase synthesizes DNA for replication while RNA polymerase synthesizes RNA for transcription by binding to DNA

Question 6

Where is upstream? Downstream?

Answer 6

upstream is near the 5’ end and downstream is near the 3’ end

Question 7

What are consensus sequences and why do bacterial promoters have them?

Answer 7

they represent the statistically most common nucleotides found at specific positions in a group of related DNA sequences
bacterial promoters must have a consensus sequence to be reliably recognized by RNA polymerase and to enable a range of gene expression levels

Question 8

What is the core enzyme of RNA polymerase? Can it recognize specific DNA sequences on its own?

Answer 8

catalytically active portion responsible for synthesizing RNA from a DNA template… can perform transcription elongation but lacks the sigma factor needed for specific promoter recognition and transcription initiation
no it can’t recognize specific DNA sequences on its own

Question 9

What is a holoenzyme?

Answer 9

Complex formed when the core enzyme binds to a sigma factor… the addition of a sigma factor allows the core enzyme to bind to promoter DNA and initiate transcription

Question 10

What does the sigma factor do? How?

Answer 10

directs the RNA polymerase to specific DNA sequences called promoters, initiating transcription by melting the -10 region of the promoter and stabilizing it as a single stranded “open complex” which then interacts with other transcription factors

Question 11

What are the general steps of transcription initiation?

Answer 11

RNA polymerase binds to DNA promoter sequence at the beginning of a gene, unwinding of double helix exposes the DNA bases, RNA polymerase can then begin synthesizing a new complementary strand of RNA with DNA as the guide

Question 12

What is the function of the sigma 70 factor in transcription initiation?

Answer 12

directs RNA polymerase to transcribe genes essential for normal cell growth and function… it binds to the core promoter RNAP and guides the holoenzyme to the promoter region of a gene, facilitates the opening of the DNA double helix to form a transcription bubble, then RNAP beings synthesizing an RNA transcript

Question 13

What does “does not dissociate” mean in relation to the sigma 70 factor in transcription initiation?

Answer 13

it means that instead of being a mandatory, one time release, the retention of a sigma 70 can lead to multiple transcription events from the same promoter, enhancing gene expression under certain conditions.
Retention of sigma 70 even after the transition to elongation

Question 14

What is the function of sigma 54 in transcription initiation?

Answer 14

sigma 54: protein the confers promoter specifically to RNAP in bacteria, but unlike sigma 70 it regulates a distinct set of genes

Question 15

What is abortive transcription?

Answer 15

process where RNA polymerase repeatedly synthesizes and releases short, non coding RNA transcripts from a DNA template, failing to produce a full length product

Question 16

What are the general steps of transcription elongation? Does it require a primer?

Answer 16

RNA polymerase moves along a DNA template strand, unwinding double helix and synthesizing a new RNA molecule by adding complementary ribonucleotides one by one in the 5’ to 3’ direction
No, RNA polymerase does not require a primer to start transcription because it can initiate synthesis directly by binding to specific DNA sequences called promoters

Question 17

What is a transcription bubble?

Answer 17

temporary, unwound region of a DNA double helix where RNA polymerase is actively creating an RNA strand during transcription
forms when enzyme opens DNA to access the template strand and moves along the DNA as transcription proceeds

Question 18

What are the substrate binding subsite and the product binding subsite?

Answer 18

Substrate binding (A) subsite is the precise location where the next correct template directed NTP binds… must be empty for the next nucleotide to be added
Product binding (P) subsite is where the 3’ end of the newly synthesized RNA chain is located after the previous nucleotide has been incorporated

Question 19

What is proofreading?

Answer 19

RNA polymerase detects a mis-incorporated nucleotide, pauses elongation, backtracks along the DNA template allowing RNA polymerases intrinsic nuclease activity to cleave the incorrect nucleotide from the 3’ end, then resume elongation

Question 20

How does supercoiling occur during elongation?

Answer 20

As RNA polymerase moves along DNA it forces the double helix to twist creating positive supercoils ahead of it and negative supercoils behind it and this must be resolved to allow transcription to continue

Question 21

How is supercoiling solved during transcription elongation?

Answer 21

topoisomerase relaxes negative supercoils
Gyrase introduces negative supercoils (resolving positive supercoils using ATP to introduce negative supercoils)

Question 22

What occurs during transcription termination?

Answer 22

the RNA polymerase stops synthesizing RNA and releases from the DNA template strand

Question 23

What is Rho-independent termination?

Answer 23

a prokaryotic mechanism that uses specific DNA sequence to signal the end of transcription without the involvement of a Rho protein

Question 24

What is the importance of the formation of stem loop structure in the exit channel?

Answer 24

a key event for Rho-independent transcription termination in bacteria, it causes the polymerase to stall then release newly synthesized RNA transcript

Question 25

What is the importance of less stable U-A hybrid helix in transcription termination?

Answer 25

these are the weak, non covalent association between RNA transcript and the DNA template, and the RNA-DNA hybrid formed in transcription is normally stable during elongation but unstable at terminator sequences to signal the end of transcription... key component of intrinsic termination and causes transcript release

Question 26

What is Rho-dependent termination?

Answer 26

a bacterial transcription termination mechanism where Rho protein (ATP powered helicase) binds to a specific RNA sequence (at the rut site), moves along the nascent RNA transcript, then unwinds the DNA-RNA duplex at a pause site to release the RNA, terminating transcription

Question 27

When is Rho-dependent termination blocked and why?

Answer 27

during gene expression to prevent premature termination Rho is prevented from loading until the end of gene or operon

Question 28

How does RNAP identify promoter sequences in the midst of huge amounts of chromosomal DNA?

Answer 28

Mobile RNAPs can explore the whole nucleoid while "searching" for promoters RNAP spends 85% of its promoter search time bound to non specific DNA

Question 29

Why is Live cell superresolution microscopy used?

Answer 29

allows scientists to observe cellular processes and structures in living cells with unprecedented detail by overcoming the diffraction limit of light enables the study of molecular dynamics within cells such as protein trafficking or cell division, under physiological conditions for extended periods