Lecture 3 and 4 Flashcards
Where can gene regulatory elements be found?
- Typically close to the transcriptional start site of prokaryotic genes
- Far upstream of the gene
- Downstream of the gene (eukaryotes)
- Within gene (introns; eukaryotes)
NtrC protein
Transcriptional activator
What does DNA looping allow the NtrC protein to do?
It allows the NtrC protein to directly interact with RNA polymerase to activate transcription from a distance in a process called “kissing”
Bacteriophage lambda
It is a virus that infects bacterial cells
How does bacteriophage lambda infect bacterial cells?
- The host chromosome is present inside the bacterial cell
- the lambda virus attaches to host cell and the lambda DNA gets injected.
- the lambda DNA cicularizes
2 possible pathways after bacteriophage lambda is inserted in the host cell
- Prophage pathway - under favorable growth conditions
- Lytic pathway - when the host cell is damaged
Prophage pathway
- Introduction of lambda DNA into the host chromosome.
- Integrated lambda DNA replicates along with host chromosome.
Lytic pathway
- Synthesis of viral proteins needed for the formation of new viruses.
- Rapid replication of lambda DNA and its packaging into complete viruses.
- Cell lysis releases a large number of new viruses.
Induction event
Damage to the host cell
Two gene regulatory proteins needed for initiating the switch between prophage and lytic pathways ?
Lambda repressor protein (cI) and Cro protein
what do the two gene regulatory proteins do?
They repress each other’s synthesis , giving rise to two states
Prophage state: Lambda repressor protein is made
Lytic state: Lambda cro protein is made
Prophage state
the lambda repressor occupies the operator
1. blocks synthesis of Cro
2. It activates its own synthesis
3. most bacteriophage DNA not transcribed
Lytic state
Cro occupies the operator
1. it blocks the synthesis of the lambda repressor
2. allows its own synthesis
3. most bacteriophage DNA is transcribed
the DNA is replicated, packaged, new bacteriophage released by host cell lysis
What triggers the switch between prophage and lytic states?
the induction event - the host’s response to DNA damage
Example of transcriptional cicuit
Prophage-lytic control
Transcriptional circuits
- Positive feedback loop - lambda repressor protein
- Negative feedback loop
- Flip-flop - Cro/lambda repressor switch
- feed forward loop
what can positive feedback loops be used to create?
Cell memory
Transcription regulator A will not be made because it is normally required for its own production.
However, there is an initial transient signal that turns on the expression of gene A.
Generations of cells after that will remember the signal.
Feed-forward loops
They decrease sensivity
Brief input does not accumulate, however prolonged input accumulated and an output is then generated.
Complex regulatory networks
Combinations of regulatory circuits in eukaryotic cells
Synthetic biology
Scientists can construct artificial circuits and examine their behavior in cells
Repressilator
A simple gene oscillator using a delayed negative feedback circuit
What was observed in the repressilator
There was increasing amplitude in the graph due to bacterial growth
Transcription attenuation
premature termination of transcription in both prokaryotes and eukaryotes
Riboswitches
Short RNA sequences that change conformation when bound by a small molecule. prokaryotes, plants, and some fungi use them to regulate gene expression.
eg. prokaryotic riboswitch that regulates purine biosynthesis
RNA transcription in prokaryotes and eukaryotes
Eukaryotes - different RNAs transcribes by different RNA polymerases
Prokaryotes - single type of RNA polymerase
prokaryotic riboswitch that regulates purine biosynthesis
Low guanine levels
- transcription of purine biosynthetic genes is on
High guanine levels
- Guanine bind riboswitch
-riboswitch undergoes conformational change
- cause rna pol to terminate transcription
- transcription of purine biosynthesis is off
Eukaryotic RNA polymerases
RNA pol I - rRNA genes
RNA pol II - mRNAs
RNA pol III - tRNAs
General transcription factors
Required for transcription initiation in eukaryotes, it helps position RNA polymerase at eukaryotic promoters.
eg: TFIID (recognizes TATA box), TFIIB, TFIIA, etc.
RNA polymerase II
- they transcribe protein-coding genes - mRNAs
- requires 5 transcription factors - TFIID, TFIIB, TFIIF, TFIIE, and TFIIH
- eukaryotic genomes lack operons
- Eukaryotic DNA is packaged into chromatin which provides an additional mode of regulation
- Eukaryotic transcriptional activity requires many gene regulatory proteins
Mediator
Intermediate between regulatory proteins and RNA polymerase
DNA looping
The mechanism by which gene regulatory proteins can act over very large distances
What do eukaryotic gene regulatory proteins function as on DNA?
Protein complexes
Coactivators and corepressors
They assemble on DNA-bound gene regulatory proteins and do not directly bind to DNA
Indirect activation of transcription
Activator proteins can alter chromatin structure and increase promoter accessibility as transcriptional machinery cannot assemble on promoters tightly packed in chromatin.
Modular design of eukaryotic activator proteins
- DNA binding domain - recognizes the specific DNA sequence
- Activation domain - accelerates frequency/rate of transcription
during mutations, biotechnology, and evolution the DBDs and ADs can be mix-matched.
Nucleosomes
Basic structure of eukaryotic chromatin.
There is DNA wound around the histone octamer (H2A, H2B, H3 and H4) x 2 + a linker H1
How do activator proteins activate transcription?
They attract, position and modify:
1. general transcription factors
2. mediators
3. RNA pol 2
either directly or indirectly
Direct activation of transcription
Activator proteins can bind directly to transcriptional machinery or the activator and attract them to promoters.
Histone code
Specific patterns of histone modification
How do nucleosomes pack as compact chromatin fibers?
- Zig zag model
- solenoid model
4 majors ways activator proteins can alter chromatin
- nucleosome sliding - using ATP + chromatin remodelling complex
- Nucleosome removal - with cooperation of histone chaperones + CRC
- Histone exchange - with cooperation of histone chaperones + CRC
- histone modifications (specific amino acids on histone tails) using writers and readers
acetylation
Addition of acetyl group
Enzyme: acetyltransferase
methylation
Addition of a methyl group
Enzyme: methyltransferase
phosphorylation
addition of phosphate group
enzyme: kinase
Writer proteins
they are histone-modifying enzymes that cause modifications on the N terminus of histone tails
Reader proteins
Recognize specific modification and provide meaning to the code
Human interferon gene regulation - histone code example
- Activator proteins binds to chromatin DNA and attract a histone acetyltransferase
- HA acetylates lysine 9 of histone H3 and lysine 8 of histone H4
- Activator protein attracts a histone kinase
- HK phosphorylates serine 10 of histone H3. ONLY after acetylation of lysine 9.
- serine modification signal the acetyltransferase to acetylated lysine. 14 of histone H3.
- TFIID and chromatin remodelling complex bind to modified histone tails and initiate transcription.
Mechanisms to inhibit transcription in eukaryotes
- competitive DNA binding
- Masking the activation surface
- Direct interaction with general TF
- recruitment of chromaitn remodelling complexes
- recruitment of histone deacetylases
- recruitment of histome methyl transferase
How do readers and writers. establish repressive form of chromatin?
- DNA methylase enzyme is attracted by Reader and methylates nearby cytosines in DNA
- DNA methyl binding proteins bind methyl groups and stabilize structure
Epigenetic inheritance
Methylation and gene expression patterns can be inherited
