18: Bacterial Transcription Flashcards
What is the RNA World Theory?
before last common ancestor, there was no DNA; RNA made proteins
Why is there are RNA step in gene expression? (3 reasons)
- The RNA step provides an amplification which contributes to differential gene expression
- Since RNA can be degraded, expression of a gene can be stopped quickly
- RNA provides additional opportunities to regulate gene expression (especially in eukaryotes during processing and export from nucleus)
What is a consensus sequence?
The most frequent base at each position in a group of functionally related DNA elements
there is a consensus sequence of all DNA/RNA binding for a protein
tight binding for most similar sequence to consensus sequence
What is a promoter?
the DNA sequence required to initiate transcription of a gene or operon
What is a terminator?
the DNA sequence required to stop transcription
What is an operon?
a set of bacterial genes transcribed from a single promoter and thus expressed from a common RNA
set of genes that are transcribed together
only bacteria have operons, but not all bacteria have operons
What is the operon structure?
- transcription start site (+1) (can be any nucleotide)
- promoter before +1 (-1 region)
- transcript (protein encoding genes)
- terminator
What are the key features of a Bacterial Promoter?
- ~100 bp
- 2 consensus sequences (highly conserved)
- -35 region and -10 region
- the rest is random bases
How were the -10 and -35 sequences found?
- comparing the sequences of many E. coli promoters
- Shared elements suggest functional importance = consensus
What are the characteristics of the -10 region?
- TA rich
- TA = less energy to separate 2HB by RNA poly
How can you test if the -35 sequence is important?
make mutations:
normal: 100%
mutate: 2%
Why are promoter seuqneces exactly idendical to consensus?
control amount of RNA transcript: less similar to consensus = less transcript = less energy waste
What is Bacterial RNA Polymerase - RNAP?
- enzyme that makes RNA transcripts using DNA as a template and nucleoside triphosphates (NTPs) as substrates
What is the RNAP structure? What are its characteristics?
- core enzyme made of 4 protein subunits that assemble and stay together (B, B’, w, a)
- can make RNA but can’t recognize promoters
- promoter specificity of RNA poly is detmined by SIGMA SUBUNIT which recognizes and binds to -35, -10 region in promoter sequence
- sigma subunit sometimes disassembles
polymerase core + sigma subunit = RNA polymerase holoenzyme
PROTEIN-PROTEIN INTERACTION
How does sigma unit recognize sequence?
fits well with promoter, stalls as it moves also DNA
What are the steps of transcription?
- initiation
- elongation
- termination
What are the steps of bacterial transcription initiation?
- RNAP holoenzyme binds to promoter (sigma subunit recognizes promoter region)
- closed complex formation of RNA poly and DNA
- RNA poly unwinds the DNA strands arounf the start site which is easy bc of the TATA box
- open complex formation + transcription bubble
What are the steps of bacterial transcription elongation?
- the first NTP is brought to the template BP with base at +1 (no primer)
- NTP as subtrates elongates in 5’-3’ direction
- sigma subunit falls off after 1st few bases so that it doesn’t interfere with transcription
- RNA poly is large and covers entire section of DNA that is unwound + DNA-RNA hybrid helix which protects ssDNA from degradation
- phosphodiester bonds form and pyrophosphates is released
- DNA is reannealing behind
What are the steps of bacterial transcription termination?
- RNA synthesis proceeds until terminator is reached and RNA polymerase falls off
- sigma rebinds RNAP and cycle repeats
- Rho independent or Rho dependent termination
What is the difference between Rho-independent/dependent termination?
INDEPENDENT:
- intrinsic termination
- depends on RNA sequence + secondary structure
- transcription of specific nucleotide sequence that leads to stem loop structure (reverse complement)
DEPENDENT:
- Rho protein binds to ssRNA and follows RNA poly as it transcripbes
- Rho catches up to RNA poly, it falls off and stops transcribing
How are different promoters transcribed at different levels?
- Some genes have better -10 and -35 sequences (not dynamic: ‘cis’ element can’t be changed by environmental factors)
- There is more than one sigma factor which recognizes different promoter sequences (specific consensus sequence for each sigma type)
- Gene specific regulatory proteins (dynamic - trans element) (negative + positive regulation transcription repressors or activators)
What protein-DNA interaction occurs in transcription?
1 alpha helix fits nicely into major groove of DNA (sequence specific)
What are the characteristics of the lac operon?
bacterial
- promoter region with a repressor sequence
- 3 proteins of lactose metabolism under 1 promoter and 1 operator
How is lactose vs glucose metabolized on bacteria?
if glucose present, this is metabolized and represses lac operon
if no glucose present and lactose is present, lac operon is active
this dynamic ensures that energy producing enzymes are not wasteful
How is lac operon expression controlled?
- Negative control: lac repressor (lacI)
- Positive control: catabolite activator protein
How is negative regulation done in lac operon?
Lac repressor: DNA binding protein
Lac operator: DNA element that binds Lac repressor
mechanism:
- lac repressor binds to operator unless a metabolite of lactose: allolactose is present
- the allolactose-Lac repressor complex does not bind DNA allowing for lactose metabolism
How is positive regulation done in lac operon?
CAP (catabolite activator protein): transcriptional activator protein
CAP binding site: DNA element that binds CAP
mechanism:
- CAP binds to a metabolite of glucose metabolism
- CAP protein can only bind to DNA in low glucose conditions causing lactose metabolism
- CAP interacts with RNA poly to help it stay in promoter region longer
What would occur to lac operon if glucose present and lactose present?
- operon off (but no repressor bc lac operon has a bad promoter, so sigma factor can’t recognize promoter without CAP)
- CAP not bound
operon off
What would occur to lac operon if glucose present and lactose absent?
- CAP not bound
- repressor bound to operator
operon off
What would occur to lac operon if glucose absent and lactose absent?
- CAP bound (low glucose)
- repressor also bound
- repression overrides activation
operon off
What would occur to lac operon if glucose absent and lactose present?
- CAP bound to promoter
- repressor off
- operon ON!
