(Dr. Heinemann) (Unit C) Topic 18 Flashcards
Central Dogma
DNA to RNA to Protein
Why is there an RNA step in gene expression?
Not required, present for more points for regulation
Reasons for an RNA step in gene expression
- Provides an amplification which contributes to differential gene expression
- RNA can be degraded, expression of a gene can be stopped quickly
- Provides additional opportunities to regulate gene expression (especially in eukaryotes)
Consensus sequence
The most frequent base at each position in a group of functionally related DNA elements
Promoter
DNA sequence required to initiate transcription of a gene or operon
Terminator
DNA sequence required to stop transcription
Operon
Set of bacterial genes trancribed from a single promoter and thus expressed from a common RNA
In bacteria, what is the transcriptional start site called?
+1 site
What are two consensus sequences in the promoter of bacteria?
-35 and -10 regions
How do we know that -35 is important?
Making mutations and seeing if it has any effect on transcription
Bacterial RNA Polymerase
RNAP
* Enzyme that makes RNA transcripts using DNA as a template and nucleoside triphosphates (NTPs) as substrates
Descibe:
RNAP core enzyme
Comprised of 4 subunits that never separate
True or False:
RNAP core enzyme can recognize promoters
False, they can only make RNA
What is promoter specificity of RNA polymerase determined by?
Sigma subunit
What is the combined polymerase core and sigma subunit known as?
RNA polymerase holoenzyme
What sequences does sigma make contact with?
-10 and -35 sequences
Initiation of Transcription:
Step 1
RNAP holoenzyme binds the promoter
* Forms closed complex
Initiation of Transcription:
Step 2
RNA polymerase unwinds the DNA strands around the start site
* Forms open complex
Open vs. Closed complex
Open complex occurs with unwinded DNA
Initiation of Transcription:
Step 3
First NTP is brought to the template base pairing with the base at +1
Are primers required for transcription?
No
Initiation of Transcription:
Step 4
Using ATP, GTP, CTP, and UTP as substrates
* Chain elongation proceeds in a 5’ - 3’ direction, following base pairing rules
What is released when phosphodiester bonds are formed?
Pyrophosphate
Initiation of Transcription:
Step 5
After addition of 5-10 nucleotides, sigma falls off the holoenzyme
Initiation of Transcription:
Step 6
Transcription bubble moves downstream (5’-3’) with the template DNA re-annealing behind
Initiation of Transcription:
Step 7
RNA synthesis proceeds until a terminator is reached and RNA polymerase falls off
Initiation of Transcription:
Step 8
Sigma rebinds RNAP and the cycle is repeated
In total, how many steps are there in initiation and replication in transcription?
8 steps
What are the 2 mechanisms for transcirption termination in prokaryotes?
- Rho-independent
- Rho-dependent
Rho-independent termination
Also known as instrinsic termination
* Terminator sequences can form loop on it self
* Pushes transcription bubble off and ends transcription
Rho-dependent transcription termination
Rho protein is attached to RNA
* Transcription slows at terminator
* Rho catches up and pushes polymerase off of DNA
What is Rho-independent transcription termination dependent on?
Sequence and Secondary structure
What is Rho-dependent transcription termination dependent on?
ATP
How are different promoters transcribed at different levels?
- Some genes have better -10 and -35 sequences
- There is more than one sigma factor (each recognizes different promoter sequences)
- Gene specific regulatory proteins
What are -10 and -35 sequences known as?
Cis element
What are gene specific regulatory proteins known as?
Trans elements
* Dynamic
Negative vs. Positive regulation
Negative
* Factors repress transcription
Positive
* Factors activate transcription
Protein - DNA interactions
One alpha helix fits nicely into the major groove of the DNA
* Sequence specific interactions
* Interactiosn with the DNA backbone
What is the preferred carbon source for E. coli?
Glucose
What happens in E. coli when glucose and lactose is present?
Cell metabolizes glucose and represses use of lactose
What is the purpose of glucose repression or catabolite repression?
Prevents wasteful duplication of energy-producing enzyme systems by repressing transcription of lac & other operons
How is transcription obscurred in lac operon?
Repressor protein blocks the promoter, preventing RNA polymerase from attaching and initiating transcription
Lac operon
* Negative control
* Positive control
- Negative: Lac repressor (LacI)
- Positive: Catabolite activator protein (CAP)
Binding sites of:
* Lac repressor
* CAP
- Lac operator: DNA element that binds Lac repressor
- CAP binding site: DNA element that binds CAP
How does Lac repressor not bind to DNA?
Allolactose - a metabolite of lactose - binds to lac repressor
* The allolactose-Lac repressor complex does not bind DNA
What allows CAP to bind to DNA?
- cAMP - A metabolite of glucose metabolism
- Low glucose conditions
