Transcription in Bacteria

Genome

the sum of all genetic infomation contained in the DNA of an organism

Transcriptome

the sum of all the RNA transcripts in a cell in a given moment (wildly varied in cell types) (think of sex cells how they vary)

Main difference between genome and transcriptome?

every cell in the body has the same genome but not every cell has the same transcriptome

Structure of DNA

forms a double helix, which is very stable

structure of RNA

can form complex secondary and tertiary structures, giving it functional capabilities

What are the types of RNA transcripts

• mRNA (messenger RNA)

• tRNA(transfer RNA)

• rRNA (ribosomal RNA)

• other non-coding RNAs

  • micro RNAs

Which type of RNA makes up about 80% of the total RNA?

rRNA (ribosomal RNA)

what is mRNA?

contains amino acid coding information for proteins

• only 3-5% of total RNA

what is tRNA?

helps in protein synthesis

• 15-20% of total RNA

what is rRNA?

helps catalyze protein synthesis reaction

builds proteins off reading DNA

• 80% of total RNA

What is transcription in a nutshell

1. RNA polymerase binds to double stranded DNA

2. DNA opens up to expose single strands

3. RNA polymerase reads template strand and adds ribonucelotides (NTPs) to growing RNA chain

4. DNA reforms double strand and exits, RNA chain exits

What is template strand?

is the one that gets matched up to and brought in the individual nucleic acids that is going to base pair with it

why is it called the coding strand instead of nontemplate?

this is due because the RNA sequence you’re building off of the DNA template is going to have the exact same sequence

what is the difference between the RNA polymerase structure between bacteria and eukaryotes

bacteria hace 1 RNA polymerase

eukaryotes have 3 (pol II is the most similar to bacteria)

What goes on inside the transcription bubble

• the polymerase reaction occurs and the growing RNA chain is paired with the template strand to form a DNA/RNA hybrid

• as RNA polymerase moves through the DNA, it rotates the DNA creating supercoils both in front and behinds it

• DNA duplex is unwound into single strands for a moving “bubble” of about 17 base pairs

what is a sigma factor?

a bacterial protein that links RNA polymerase to the transcriptional start site at the front of a gene

• equivalent of transcription factors

• sigma factors control which genes get transcribed and when

what do sigma factors do?

they help RNA polymerase know where the start of a gene is

they help RNA polymerase know which genes to transcribe

What are promoters

region of RNA at the start of a gene, whose sequence determines where transcription begins and when

• region where RNA polymerase binds (around 100bp, -70 to +30)

promoter regions consensus sequence

region in the promoter where the sigma factor binds, -35 and -10 regions both contain consensus sequences

upstream promoter (UP) element

AT rich sequence, recognized by a subunit of RNA polymerase. Increases binding and transcription

what do promoters help do?

helps sigma factors know where to bind on DNA

Footprinting Assay

binds proteins (ex Sigma factors) to DNA, then chop up the DNA, and see which regions are protected

Nucleolytic proofreading

polymerase backtracks and nuclease activity hydrolyzes bond upstream of mismatched base

kinetic proofreading

detects (by stalling) mismatches immediately after phosphodiester bond formation

reverses the catalytic reaction (pyrophosphorolysis)

Rho-Independent

Requires two sequences in the RNA

• Termination sequence in RNA about 15-20 nucleotides end of RNA

  • creates hairpin structure

• AAA in template strand creates UUU in RNA strand

  • hairpin dislodges RNA at AAA/UUU interface

Rho-Dependent

rut site- rho utilization site sequence on RNA that recruits rho

⍴(rho) helices- helices that travels along RNA transcript

when ⍴ encounters polymerase, will hydrolyze ATP to dislodge transcript and shut down transcription

Transcription initiation, part 1

• RNA polymerase holoenzyme (core+sigma factor) binds to DNA at promoter consensus sequence. this complex is initially closed

• structural change then causes the DNA to melt (separates into 2 strands) and enters the polymerase active site, creating a transcription-competent open complex

this process is spontaneous, reversible, and requires no energy

Transcription initiation, Part 2

RNA polymerase does not require a primer

NTPs are added quickly, but the structure is unstable for the first 8-10dp and will spontaneously stop and leave the channel. This is abortive initiation

abortive initiation occurs frequently, if so, the process begins again until about 10 bases are added, stabilizing the transcript

Transcription initiation part 3

Sigma factor is initially blocking the exit channel for RNA. The growing transcript will dislodge sigma factor form the exit channel, causing sigma factor to release from the polymerase

once released the complex exits the promotor and initiation is complete

→ this is promoter clearance

Transcription Elongation

3 channels in the polymerase

• DNA entry channel

• NTP entry channel

• RNA exit channel

• (no DNA exit channel)

a pin region of the polymerase helps to keep the DNA strands separate while in the “bubble”

foot printing assay

shows the areas where protein can bind to in DNA