(2) Chapter 8: RNA Synthesis and Processing Flashcards
Bacterial RNA polymerase
- catalyzes polymerization of NTPs as directed by a DNA template (in 5-3 direction).
- has 5 subunits: alpha, beta, omega, sigma,
NTPs
ribonucleoside 5’ triphosphate
How does transcription initiate in prokaryote cells?
de novo, no primer is needed. (promoters are used)
Alpha subunit of bacterial RNA polymerase
- identifies the correct sites for transcription initiation
- most bacteria have many alpha subunits
- binds to both -10 and -35 regions
Promoter
- gene sequence to which RNA polymerase binds to initiate transcription
- 6 nucleotides long
- located at 10 and 35 bp’s upstream of transcription start site.
consensus sequences
- bases most frequently found in different promoters
- genes that have promoters that differ from consensus sequences are transcribed less efficiently
Bacterial DNA unwinding mechanism
- polymerase unwinds 12-14 bases of DNA then releases sigma group to form an open-promoter complex for transcription
- polymerase maintains an unwound region of about 15 bps at a time
When is the sigma subunit released from polymerase?
after addition of about 10 nucleotides during transcription.
Prokaryote transcription termination
-When polymerase encounters a STOP Signal, transcription is terminated
What is the most common stop signal in prokaryote transcription? How does it work?
- GC-rich sequence followed by seven A residues.
- transcription of this symmetrical inverted repeat sequence results in a RNA segment that forms a stable-loop structure
- loop distrupts DNA template association
Enzymes involved in metabolism of lactose
- beta galactosidase
- lactose permease
- transacetylase
beta-galactosidase
-cleaves lactose into glucose and galactose
lactose permease
transports lactose into the cell
transacetylase
inactivates toxic thiogalactosides that are transported into the cell along with lactose
Operon
a single unit that holds genes that encode specific enzymes
operator in prokaryote transcription
- a loci that controls transcription
- adjacent to transcription initiation site
- part of operon
repressor in prokaryote transcription
- “i loci”; a loci that controls transcription.
- encodes a protein that binds to the operator
- blocks transcription when bound to operator
How does the lactose operon work?
when lactose is present in normal cells, it binds to the repressor (i loci) and prevents the repressor from binding to the operator; so, genes are expressed
How is control of transcription mediated?
-the interaction of regulatory proteins with specific DNA sequences.
Cis-acting control elements
-affect expression of linked genes on the same DNA molecule (the operator)
Negative control of transcription
The regulatory protein (the repressor) blocks transcription. Ex) lac operon
Positive control of transcription
Regulatory proteins activate transcription
Ex) E.coli
Explain the process of positive control of the lac operon by glucose (prokaryote)
- If glucose is present: lac operon is repressed, even if lactose is also present
- if glucose levels decrease, cAMP levels increase
- cAMP binds to CAP
- CAP binds to target DNA sequence which is upstream lac operon
- CAP facilitated binding of RNA polymerase to the promoter
CAP
catabolite activator protein (regulatory protein). active in prokaryote cells
How many polymerases are involved in eukaryotic transcription?Are they complex?
-three RNA polymerases transcribe different classes of genes. all are very complex and consist of 12-17 substrates
RNA polymerase II
synthesizes mRNA, requires initiation factors that are not associated with polymerase
General transcription factors
- proteins involved in transcription from polymerase II promoters
- very important
What percentage of genes in the human ganome encode transcription factors?
10%
TATA box
- a sequence element found in a promoter
- 10 sequence of bacterial promoters
Inr promoter elements
initiator elements, sometimes found as a promoter
TFIIB promoter elements
recognition elements, ex. BRE
downstream promoter elements
DCE, MTE, DPE
TFIID transcription factor
composed of multiple subunits
- TATA binding protein (TBP)
- TAFs that bind to the Inr, DCE, MTE, and DPE sequences
formation of polymerase II Preinitiation complex
- has five general transcription factors
1. TATA binding protein with other subunits
2. TFIIB
3. TFIIF
4. TFIIE
5. TFIIH
Mediator
- a protein complex of more than 20 subunits
- binds to nonphosphorylated CTD when transcription initiates
- released following phosphorylation of CTD when transcription initiates
After transcription is initiated (CTD has been phosphorylated by the mediator) what happens to the phosphorylated CTD?
The mediator complex is released.
-the phosphorylated CTD binds elongation and processing factors that facilitate mRNA synthesis & processing
What is the function of RNA polymerase 1 in eukaryotic cells in eukaryotic cells?
transcribes rRNA genes
What are the end products of transcription by RNA polymerase 1 in eukaryotic cells?
45S pre-rRNA: processed to yield 28S, 18S, 5.8S rRNAs
What two transcription factors recruit RNA polymerase 1 to form an initiation complex in eukaryotic cells?
- UBF (upstream binding factor)
- SL 1 (selectivity factor 1)
Polymerase III (eukaryotic)
- transcribes genes for tRNAs, 5S rRNA, and some small RNAs
- expressed from 3 types of promoters.
Gene transfer assay
-identify regulatory sequences of expression in eukaryotic genes
Regulatory sequence
Directs expression of the reporter gene in cultured cells, ligated interaction to reporter gene
Reporter gene
Encodes an easily detectable enzyme (fluorescent structure)
Cis-acting regulatory sequences
Located upstream of transcription start site
What are the cis-acting regulatory sequences that encode thymidine kinase in the herpes simplex virus?
- TATA
2. GC boxes
Enhancer
- regulatory sequences located farther away from the start site.
- usually have multiple sequence elements that bind different regulatory proteins that all work together to regulate gene expression
- activity doesn’t depend on their distance from, or orientation with respect to the transcription initiation site
- activity is epecific for the promoter of its target gene
How do enhancers function to regulate transcription in eukaryotes?
-function by binding transcription factors that then regulate RNA polymerase
DNA looping
-allows transcription factors bound to a distant enhancer to interact with proteins associated with the RNA polymerase/mediator complex at the promoter
In B lymphocytes, what controls transcription of immunoglobin genes?
An enhancer that is active in lymphocytes, but not other cell types
The regulatory sequence in B lymphocytes is partly responsible for what kind of expression of the immunoglobin?
Tissue specific
Immunogobin heavy-chain enhancer
- has at least nine distinct sequence elements that serve as protein binding sites.
- contains positive & negative regulatory elements
Positive regulatory element
Activates transcription in B lymphocytes
Negative regulatory elements
Inhibits transcription in other cell types
How is specificity maintained between interaction of enhancers and promoters?
-by insulators or barrier elements
Insulators/barrier elements
Divide chromosomes into independent domains and prevent enhancers from acting on promoters located in an adjacent domain.
Electrophoretic-mobility shift assay
radiolabled DNA fragments are incubated with a protein and then subjected to electrophoresis in a non-denaturing gel. Migration of DNA fragment is slowed by a bound protein