Transcription and Transcriptional Regulation Flashcards
What are some uses of PCR?
- detection of infectious organisms – such as microbes and viruses
- forensics and disease diagnosis
- detection of gene expression (RT-PCR)
- protein engineering and DNA analysis
What determines the ends of the product of PCR?
The 5’ ends of the primer used
Northern blotting steps
- isolate total RNA
- denature and separate RNA
by electrophoresis - transfer to membrane
- Detect using labeled (radioactive)
antisense probe to hybridize
TBP:
TATA-box Binding Protein, eukaryotes
What does TFIIH do?
acts as both a DNA helicase and a kinase
during initiation
Does an RNA strand have to be completely made before a new one is started?
No. The synthesis of additional RNA molecules can be started before previous RNA molecules are completed
Does RNA polymerase need a primer?
No.
What is the proofreading mechanism of RNA polymerase?
If an incorrect nucleotide is added to the growing RNA chain, the polymerase can back up, and the active site of the enzyme can perform an excision reaction that resembles the reverse of the polymerization reaction, except that a water molecule replaces the pyrophosphate and a nucleoside monophosphate is released.
snRNAs
small nuclear RNAs, function in a variety of nuclear processes, including the splicing of pre-mRNA
What is the sigma factor?
a subunit that associates with the RNA core enzyme and assists in beginning DNA transcription in prokaryotes. bind specific promoter elements
transcription bubble
the region of unpaired DNA which is stabilized by the binding of the sigma factor to unpaired bases on one of the exposed strands
How is transcription terminated in prokaryotes?
string of A-T nucleotide pairs which fold into a hairpin. The formation of the hairpin helps to disengage the RNA transcript from the active site.
Are all termination sequences the same?
no, there is variation, as long as they form hairpins its chill
If an RNA sequence is 5’-GUGUACACAU-3’, what is the sequence of its DNA template?
5’-ATGTGTACAC-3’
The strand in dsDNA used as a template for RNA synthesis depends on:
the orientation of the promoter
The majority of RNA transcripts in cells are _____ and predominately _____
The majority of RNA transcripts in cells are noncoding RNAs and predominately rRNA
Which of the eukaryotic RNA polymerases is responsible for mRNA synthesis?
RNA polymerase II
Which is more accurate, DNA or RNA polymerase?
DNA polymerase
Which is more processive, DNA or RNA polymerase?
RNA polymerase
Genes that are expressed in practically all cells in the human body include:
tRNAs
RNA polymerase
histones
ribosomal proteins
Restriction nucleases cut
DNA at specific sequences
Main differences between RNA polymerase II and bacterial RNA polymerase
1) While bacterial RNA polymerase requires only a single transcription initiation factor (sigma) to begin transcription, eukaryotic RNA polymerases require many such factors, collectively called the general trasncription factors
2) Eukaryotic transcription initiation must take place on DNA that is packaged into nucleosomes and higher-order forms of chromatin structure, features that are absent from bacterial chromosomes
How far is the TATA box from the transcription start site?
What binds to it?
About 25 nucleosides upstream from the transcription start site
TFIID
What are transcriptional activators?
gene regulatory proteins that bind to specific DNA sequences to help attract RNA polymerase II to the start point of transcription
Where do transcriptional activators bind?
enhancers
What is a mediator?
a large protein complex which allows the activator proteins to communicate properly with the polymerase II and the general transcription factors.
What is the last step of transcription initiation?
the recruitment of chromatin-modifying enzymes
Steps at which eukaryotic gene expression can be controlled
1) transcriptional control
2) RNA processing control
3) RNA transport and localization control
4) translation control
5) mRNA degradation control
6) protein activity control
What do restriction nucleases do?
facilitate isolation and manipulation of DNA at specific sites
How does vector cloning work?
restriction endonuclease is added to cleave DNA and then the DNA fragment to be cloned is incorporated into the strand/
DNA library
a collection of cloned plasmid molecules
reverse transcriptase
makes DNA from mRNA
Order of binding for prokaryotes
1) TBP binds to TATA box
2) TFIIB binds to complex
3) Pol II binds to complex
4) TFIIH binds to complex, acts as both a DNA helicase and a kinase during initiation
5) release of general factors except TBP and Pol II, C-terminus of Pol II becomes phosphorylated.
what does a gene consist of?
promoter and transcription unit
What do repressors bind?
silencers
prokaryotic vs eukaryotic location of binding sites
Prokaryotic: typically close to the promoter
Eukaryotic: can be far or close, upstream, downstream, in the transcription unit itself
What does it mean for something to be a cis-regulatory sequence?
“Cis”: the DNA element is on the same piece of DNA as the gene promoter it regulates
How do proteins recognize specific DNA sequences?
By recognizing H-bond donors, acceptors, or methyl groups (specificity)
Which contacts are likely important for the affinity of DNA binding proteins?
contacts between the binding protein and
1) DNA phosphates
2) DNA deoxyriboses
3) DNA bases
What activates CAP in the lac operon?
cAMP
How does lactose activate lac operon transcription?
1) lactose becomes allolactose
2) allolactose then activates lac operon transcription by activating the lac repressor
How is glucose related to adenynyl cyclase?
Glucose inhibits the activity of adenylyl cyclase
What does adenynyl cyclase do?
ATP —> cAMP
When is the lac operon transcribed?
lac operon is only transcribed when lactose is present and glucose is not
cis-regulatory elements vs promoters
cis-regulatory elements are specific sequences bound by transcriptional regulators,
promoters are common between genes and are where general transcription factors and RNA polymerase binds
how do eukaryotic transcriptional regulators act?
They act indirectly to affect RNA polymerase-promoter association
where would an enhancer bind?
cis-regulatory sequence
What complexes formed on transcription regulators act as activators?
histone modifiers (acetyltransferases, kinases, methyltransferases) and histone remodelers
What complexes formed on transcription regulators act as modifiers?
deacetylases, methyltransferases
How are regulators regulated?
1) expression levels
2) activity levels
3) localization
examples of reporter genes
epitope-tagged proteins (HA; Western)
luciferin (light emission when treated with luciferase)
GFP or GFP-tagged proteins (fluoresce green under UV light)
what can gene expression constructs be used for?
Can be used to express:
1) a protein or RNA of interest (ex. HAtagged
Upf1)
2) a reporter gene controlled in expression by an element of interest
(gene product is easily measured to “report” on transcriptional activity)
