Lecture 24 Flashcards
Transcription
- the process of making RNA from a DNA template
- highly selective
- RNA polymerase joins nucleotide together using one strand of a double stranded DNA molecule as a template
- does not require a primer
- requires ribonucleoside triphosphate forms - extra two phosphates are removed as nucleotide is added to the chain
- proceeds in 5’ to 3’ direction
What is meant by transcription is a highly selective process?
only a small part of the DNA is transcribed
polycistronic
one mRNA is produced from the RNA coding region, but this mRNA contains the information for more than one gene product
Prokaryotic mRNA
- polycistronic
- usually no introns
- contain conserved sequences that are upstream and are important regions of the promoter
- one RNA polymerase (different from the primase of replication)
Eukaryotic mRNA
- usually monocistronic
- contains introns
- contain conserved sequences that are upstream and are important regions of the promoter
- 3 different RNA polymerases with their own promoter elements and transcribing specific RNA types
introns
intervening sequences that are removed from the initial RNA transcript prior to translation
monocistronic
the mRNA codes for only one gene
upstream
prior to the start of transcription
Types of RNA in all cells
mRNA
rRNA
tRNA
Types on RNA in only eukaryotes
Pre-messenger RNA (pre-mRNA) small nuclear RNA (snRNA) small nucleolar RNA (snoRNA) small interfering RNA (siRNA) piwi-interacting RNA
Types of RNA in only prokaryotes
crRNA
pre-mRNA
the initial transcript of mRNA in eukaryotes
processed as it is converted into its active mRNA form
template strand
only one strand of DNA is used for transcription - this is that strand
nontemplate strand
sense strand or coding strand
the strand not used at the template for transcription
RNA is ___ and ___ to the template strand
antiparallel
complementary
New nucleotide on RNA are laid down in the ___ direction
5’ to 3’
The template strand must run ___ in the direction of transcription
3’ to 5’
transcription unit
segment of DNA that codes for an RNA molecule and the sequences necessary for its transcription
coding strand
nontemplate strand
sense strand
nontemplate strand
promoter
sequence of DNA that contains important controlling regions for transcription
where RNA polymerase binds to the DNA
usually contain a consensus sequence
Where does RNA polymerase bind to DNA for transcription?
the promoter
downstream
from the start of transcription toward the end of the RNA coding region
teminator
a series of sequences that cause termination of transcription to occur
steps of transcription
- two strands of DNA separate
- various enzymes and proteins bind to the DNA and molecules come to together to make RNA
- RNA polymerase does not require a primer to initiate RNA synthesis
- RNA nucleotides are laid down antiparallel and complementary to the template strand from the 3’ end in a 5’ to 3’ manner
- as transcription proceeds, RNA is displaced from the template strand allowing DNA to come back together
What joins the nucleotides together in transcription
phosphodiester bonds
Describe the units of the holoenzyme of RNA polymerase in prokaryotes
two alpha subunits one beta subunit one beta prime subunit one omega subunit one sigma subunit
What is the difference between holoenzyme and core enzyme of RNA polymerase in prokaryotes?
the sigma subunit is not in the core enzyme
4 stages of transcription in prokaryotes
- template binding
- chain initiation
- chain elongation
- chain termination
What is the outcome of the sigma subunit not being present? pro
A core enzyme will bind DNA and allow initiation but will not occur at the right place without sigma
Which form of the enzyme is required for which stage of transcription
pro
the holoenzyme is required for binding and initiation
after initiation the sigma subunit dissociates leaving the core enzyme to complete elongation and termination
At what nucleotide does transcription start? pro
+1 nucleotide
what are the two important consensus sequences in the promoter
pro
pribnow box
-35 control region
pribnow box
pro
located in the promoter
-10 region
5’-TATAAT-3’
-35 control region
pro
located in the promoter
5’TTGACA-3’
consensus sequence
pro
a sequence that describes the nucleotides most often present in a segment of interest
given as the sequenced on the coding strand, not template strand
implies important function for the sequence
Describe binding
pro
- holoenzyme binds to the promote to form a closed complex, this requires the proper -10 and -35 regions
the DNA strands separate to form and open complex or transcription bubble
Describe initiation
pro
- starts at +1 nucleotide
- sigma is still required for initiation to begin at correct place
- polymerase changes shape and can no longer bind the promoter allowing it to move downstream
- sigma stays attached until about 9-12 nucleotides are joined and then dissociates
transcription bubble
pro
short stretch of unwound DNA about 18 nts long
has positive supercoiling ahead and negative behind
What leads to hairpin formation on the RNA during transcription?
pro
inverted repeats
this is important to transcription termination
Rho-independent termination
pro
inverted repeat on the DNA near where termination should occur
they are transcribed leading to a hairpin formation which causes RNA polymerase to pause
just after the inverted repeat area there are adenine residues which are transcribed as a string of uracil residues
the weak interaction between a and u causes the RNA to dissociate from the DNA
Rho-dependent termination
pro
- the inverted repeats and adenine residue are still present and transcribed
- Rho binds to the RNA and moves toward the 3’ end
- RNA polymerase pauses at hairpin
- helicase activity of Rho causes the RNA-DNA hybrid to unwind and dissociate
Shine-Dalgarno sequence
pro
- within the leader region of prokaryotic mRNA
- important for proper binding of the ribosome in translation
- 7 nts upstream
leader region of prokaryotic mRNA
5’untranslated region
RNA polymerases in eukaryotes
I
II
III
eukaryotic RNA polymerase I transcribes…
large rRNA
eukaryotic RNA polymerase II transcribes…
mRNA
most snRNA
snoRNA
miRNA
eukaryotic RNA polymerase III transcribes…
small rRNA
tRNA
some snRNA
____ processing occurs in eukaryotes but not prokaryotes
mRNA
terms to describe the initial transcript of mRNA before processing
- hnRNA
- pre-mRNA
- primary transcript for mRNA
large rRNA
18s
5.8S
28S
rRNA genes are transcribed into…
1 pre-rRNA molecule that undergoes processing to release the 18S, 5.8S, and 28S rRNA
rRNA transcription units
- rRNA genes are transcribed as a unit with spacer sequences between the final RNA molecules
- methylation occurs in the areas that will become the final rRNA and the spacers are removed to release the final products
- there are multiple copies of transcription units in a eukaryotic cell
spacer sequences
- found in rRNA transcription units
- they contain control regions, promoters, and terminators
- they separate different rRNA molecules
cis elements
- nucleotide sequences that are close to the coding region of a gene
- help the cell determine when the gene should be transcribed
- binding sites for proteins or RNAS
- used in the production of mRNA
trans-acting factors
- proteins or RNAs from other genes
- attach to cis-elements or other transcription factors
- recruit RNA polymerase
- used in the production of mRNA
describe the eukaryotic promoter
- has two regions: he core promoter and the regulatory promoter
TATA box
- near -25
- consensus sequence read off non-template strand
- part of the core promoter
- important to the initiation of tx of mRNA
regions of the eukaryotic regulatory promoter
CAAT box
GC box
Octamer box
CAAT box
- near -80
- important to initiation of tx
GC box
- may have more than 1 and the location varies
- helps RNA polymerase bind near start site of tx
Octamer box
- location varies
- helps RNA polymerase bind properly in initiation
enhancers
- not in the promoterbut on the same piece of DNA and often far away from the gene they influence
- cis acting
- required for maximum transcription of eukaryotic genes
transcription activator proteins
bind enhancers and help them to interact with transcription factors near the start of transcription
core promoter
- basal promoter
- immediately upstream of gene
- where the basal transcription apparatus binds
- has TATA box and other recognition sequences important to binding transcription factors
regulatory promoter
- immediately upstream of core promoter
- variety of consensus sequences
basal transcription apparatus
general transcription factors that assemble near the start site and initiate minimal transcription
binds to the core promoter
prokaryotic initiation of transcription
- compare sigma subunit to hound dog sniffing out proper starting location
eukaryotic initiation of transcription
- compare initiation factors to a siren calling or recruiting RNA polymerases so the enzyme binds properly
assembling of transcription factors, polymerase, and other proteins to allow transcription initiation in eukaryotes
- orderly process
1. transcription factor IID binds to TATA box
2. other transcription factors and RNA polymerase II can now bind to the core promotor
3. transcription activator proteins bind to enhancers
4. DNA loops out to allow the proteins bound to enhancers to interact with the main transcription complex
5. transcriptional activator protein binds to sequences in regulator promoter to interactors with the basal transcription apparatus
6. conformation changes separate DNA strands and place template strand in active site of RNA polymerase
7. RNA polymerase can now add nucleotide to produce RNA
eukaryotic elongation
- after ~30bp are added, RNA polymerase leaves the promotor to begin elongation
- many transcription factor stay bound to promotor to reinitiate transcription
- the structure of RNA polymerase causes separation of the newly formed RNA strand from the template strand
size of eukaryotic transcription bubble
about 8bp of DNA-RNA hybrid
RNA polymerase II structure
- has own helicase activity to separate DNA strands
- site for each DNA strands so they can stay separate and so RNA can be produced antiparallel ad complementary to the template
Ratl exonuclease
required for termination of polymerase II transcription in eukaryotes
describe eukaryotic termination
mRNA is cleaved at consensus sequence leaving an RNA tail complexed to DNA
Ratl exonuclease then bind the RNA and degrades the trailing RNA pieces from 5’ to 3’
3 types of mRNA processing
- removal of introns
- PolyA tail added to 3’ end
- capping of 5’ end
capping
- part of processing pre-mRNA to form mRNA
- the 5’ end is capped with 7-methyl guanosine triphosphate in a 5’ to 5’ linkage
- 5’ phosphate end is removed and the 7-methyl GTP is added in 5’ to 5’ manner resulting in three phosphates between the 7-methyl guanosine and the end of the pre-mRNA
poly-A tail
- part of processing pre-mRNA to form mRNA
- done after pre-mRNA is released form RNA polymerase II
- pre-mRNA is cleaved near 3’ end and series of adenine residues is added
intron removal
- part of processing pre-mRNA to form mRNA
- introns removed and exons joined together
RNA polymerase III
transcribes small RNAs such as tRNA, 55 rRNA, mostsnRNAs
initiation with RNA polymerase III
- the consensus sequence for their transcripts are found within the gene for the RNA
- transcription factors interact with the internal promoter elements and with polymerase III to position it for proper initiation at +1 site
prokaryote vs eukaryote transcription and processing
- in eukaryote transcription and processing occurs in the nucleus but translation in the cytoplas - in prokaryotes there is no nucleus
- no processing required in prokaryotes
- eukaryotes have different polymerases for different types of RNA
- eukaryotic mRNAs are monocistronic - prokaryotic mRNA are polycistronic
