Transcription and Translation 1 (lecture 7) Flashcards
What are the major differences between DNA and RNA
- RNA is single stranded
- RNA has ribonucleotides instead of deoxyribonucleotides
- RNA has Uracil instead of Thymine
- RNA can fold into complex 3-dimensional structures allowing some RNAs to have precise structural and catalytic functions
Function of snRNAs
function in a variety of nuclear processes, including the splicing of pre-mRNA
function of snoRNAS
small nucleolar RNAs, help to process and chemically modify rRNAs
function of miRNAs
Micro RNAs, regulate gene expression by blocking translation of specific mRNAs and cause their degradation
Function of siRNAs
small interfering RNAs, turn off gene expression by directing the degradation of selective mRNAs and the establishment of compact chromatin structures
mRNA make up _____% of total cellular RNA
3-5%
do many genes have RNA as their final product
yes
non-coding RNA serve as ____ and ____ components for many biological processes
enzymatic and structural
Direct the splicing of pre-mRNA to form mRNA
snRNA
regulate eukaryotic gene expression by degrading select mRNA
small interfering RNA (siRNA)
Regulate gene expression by blocking translation of selective mRNA
micro RNA (miRNA)
Characteristics of prokaryotic RNA polymerase
- Bacterial RNA polymerase (RNA Polymerase)- multi subunit complex
- The holoenzyme is alpha2betabeta’sigma
- Catalyzes the formation of phosphodiester bonds that link nucleotides
- RNA polymerase has proof-reading nuclease activity
- Lower fidelity of RNA synthesis can be tolerate
RNA transcription uses what strand
Template strand
what is the most important and most regulated step in gene expression
Initiation
The sigma subunit binds to the promotor to form an
open promoter complex
RNA polymerase assembles into holoenzyme _______
alpha2betabeta’sigma
sigma subunit binds to the promotor and unwinds a short ___BP segment of double stranded DNA to form a transcription bubble
17
When transcription begins a short RNA is formed via a_____ mechanism. The stress that is caused with polymerase still bound to promoter pulls DNA into active site causes short RNA to
scrunching mechanism, be released by abortive initiation
_____ of nucleoside triphosphate provides energy to form phosphodiester bonds and drive RNA polymerase forward
Hydrolysis
in prokaryotes two common promoter motifs are known as the ____ sequence and the ________ are present on 5’ upstream side of the transcription start site
-35 sequence (TTGACA) -10 sequence (TATAAT)
the sigma factor is released during what and reattached when
Sigma is released after promoter clearance, right before elongation. It is then reattached after termination
Elongation phase begins after the formation of
nascent RAN with about 10 nucleotides
Elongation generates superhelical tension that is eased by
DNA gyrase
Transcription continues till RNA polymerase meets
termination signals
Termination signals
- Palindromic GC rich region followed by AT rich region
- RNA transcript of this region is self complementary
- forms a stable hairpin structure with a stem and a loop (due to high GC content)
- This sequence followed by a poly(U) tail
what happens when RNA polymerase encounters stable hairpin loop
- RNA polymerase pauses when it encounters this loop and formation of phosphodiester bonds stops
- DNA-RNA hybrid destabilized due to rU-dA bonds (weak) and dissociates
- Hairpin structure destabilizes the interaction of RNA with RNA polymerase and RNA dissociates
- Unwound Region of DNA rewinds with its partner to form DNA duplex
- Transcription bubble closes
In prokaryotes, all RNA molecules are synthesized by a single RNA polymerase while in eukaryotes there are ____ RNA polymerases.
3
What RNA polymerase transcribes all protein-coding gens, plus snoRNA gens, miRNA genes, siRNA genes, INCRNA genes, and most snRNA genes in eukaryotes
RNA polymerase II
What RNA polymerase transcribes tRNA genes, 5S rRNA genes, some snRNA genes, and genes for other small RNAs
RNA polymerase III
Eukaryotic RNA polymerase II requires additional proteins during transcription these are known as
general transcription factors
What are the unique features of RNA polymerase II
- RNA polymerase II contains a unique carboxyl-terminal domain of the 220-kD subunit called CTD
- The activity of RNA polymerase II is regulated by phosphorylation mainly on Ser residues of the CTD
The general transcription factors for RNA polymerase II are denoted as
TFIIA, TFIIB,TFIIC, TFIID
The general transcription factors for RNA polymerase II assemble at the _____ before ___
at the promoter before transcription
Functions of general transcription factors for RNA polymerase II
- help to position the RNA polymerase correctly at the promoter
- Aid in pulling apart the two strands of DNA to allow transcription to begin
- release RNA polymerase from the promoter into the elongation mode once transcription has begun
Eukaryotic transcription begins with binding of ____ to the _____
TFIID to the TATA box
what is the subunit of TFIID that recognizes TATA called
TATA-box binding protein (TBP)
After TFIID is bound to TATA box ____ is recruited. Followed by ___, ____, ____ and ____. When all of these factors are joined they form the _____
TFIIB, TFIIF, RNA Polymerase II, TFIIE, and TFIIH, Transcription initiation complex
What transcription factor is directly on RNA polymerase II
TFIIF
what part of the transcription initiation complex unwinds the DNA double helix and exposes the DNA template strand.
TFIIH
_________ of _____ by _____, causes RNA polymerase II to leave the promoter and begin elongation
phosphorylation of CTD by TFIIH
what are the subunits of TFIID and their functions
- TBP: recognizes TATA box
- TAF: Recognizes other DNA sequences near the transcription start point; regulates DNA-binding by TBP
Function of TFIIB
Recognizes BRE element in promoters; accurately positions RNA polymerase at the start site of transcription
Function of TFIIF
Stabilizes RNA polymerase interaction with TBP and TFIIB; helps attract TFIIE and TFIIH
Function of TFIIE
Attracts and regulates TFIIH
Function of TFIIH
Unwinds DNA at the transcription start point, phosphorylates Ser5 of the RNA polymerase CTD; releases RNA polymerase from the promoter
____ box is the most common promoter sequence but some additional regualtory sequences are
TATA box, CAAT box, or GC box
RNA polymerase II creates a superhelical tension as it moves along the eukaryotic DNA but this tension is removed by
DNA topoisomerase
For a DNA molecule with one free end 0r a nick in one strand that serves as a swivel the DNA double helix rates by one turn every ____ nucleotide pairs opened. Since DNA helix does not have a free end it relieves the superhelical tension by bending into 1 ______ every ____ nucleotide pairs opened
10, supercoiled loops every 10 nucleotide pairs opened
What is the difference between positive and negative supercoiling
Negative supercoils have the opposite handedness form the positive supercoils that form when a region of DNA helix opens. (note whenever a region of the helix opens it removes these negative supercoils from bacterial DNA, reducing the superhelical tension)
Eukaryotic DNA i packaged into ______, which are arranged in higher order chromatin structures
Nucleosomes
_____, a protein complex, allows the activator proteins to communicate with RNA Polymerase II and the general transcription factors
Mediator
Chromatin modifying enzymes are recruited to provide greater access to DNA
- Chromatin remodeling complexes
- Histone modifying enzymes
Transcription elongation in eukaryotes is tightly coupled to ____ processing
RNA
Immediate product of RNA polymerase II is _____ or ____
pre-mRNA or primary transcript
Eukaryotic mRNA needs to be _____
processed
____ modifications to mRNA are introduced to allow the cell to assess whether both ends of an mRNA molecule are present (message is intact) before it is exported form the nucleus to cytosol for translation
Covalent Modifications
How is the RNA processed before translation in eukaryotes
- Modification of the 5’ end of mRNA (RNA capping)
- Removal of non-coding sequences (RNA splicing)
- Modification of the 3’ end of mRNA (polyadenylation)
The ___ end of the nascent RNA molecule is modified by addition of a cap
5’ end
what is the structure of the 5’ RNA cap
7-methylguanosine joined to the 5’ end of RNA via a 5’-5’ triphosphate bridge.
What is the role of the 5’ RNA cap
- Helps to distinguish mRNA from other RNAs
- Define The translation start site
- Stabilizes mRNA by protecting their 5’ ends form phosphates and nucleases
- Binds a protein complex CBC (Cap binding complex)- helps in RNA processing and export
Eukaryotic genes are discontinuous because they are composed of ___ and ___
exons and introns
The _____ must be excised and _____ linked to form the final mRNA (this process is called ____)
introns, exons, splicing
mRNA splicing is carried out by _____ (assemblies of protein and small nuclear RNA)
Spliceosomes
explain polyadenylation of 3’ end of pre-mRNA
- The 3’ end of an mRNA is specified by a signal in DNA
- This signal is transcribed into mRNA
- These unique consensus sequences are AAUAAA, GU or U rich sequences and CA
- recognized by RNA binding proteins and RNA processing enzymes
- Two of the multisubunit proteins, called CstF (cleavage stimulation factor) and CPSF (cleavage and polyadenylation specificity factor) are of special importance
- the two proteins travel the RNA polymerase tail and are transferred to the 3’ end processing sequence on the RNA molecule as it emerges from the RNA polymerase
- The AAUAAA at 3’ end of RNA is bound by cleavage and polyadenylation factor (CPSF)
- The GU rich element beyond the cleavage site is bound by cleavage stimulation factor (CstF)
- CA sequence bound by a third factor
- RNA is cleaved at the CA sequence by endonuclease
- poly A polymerase (PAP) adds 200 A nucleotides to the 3’ end produced by the cleavage (from ATP) (note PAP does not require a template)
- poly a binding proteins assemble onto the poly-a tail
Bacterial mRNAs are ____, whereas eukaryotic mRNAs are mostly _____
polycistronic, monocistronic
The 5’ and 3’ ends of bacterial mRNAs are not _____ while eukaryotic mRNA
modified, has a 5’ cap and a 3’ poly A tail
Do bacterial mRNAs have introns
no ( but eukaryotes due so they must be removed by splicing)
