Lecture 7 Flashcards
Genetic instruction is a ____ letter alphabet
4
Where is hereditary information stored?
In DNA
What directs the formation of an organism?
4 nucleotides
What do nucleotides specify?
The linear order of amino acids in each protein
What determines the biological functions within a cell?
Properties of proteins
How does DNA control protein synthesis?
DNA does not control protein synthesis directly; uses RNA as an intermediary
What is copied into RNA? What is the name of the process?
A gene (appropriate piece of DNA); Transcription
RNA is used as a template to direct the synthesis of a protein during what process?
Translation
What is the central dogma of molecular biology?
DNA(replication)DNA—transcription—>RNA—-translation—>Protein
What are the similarities of RNA and DNA?
Both are linear polymers
Made of nucleotides connected by phosphodiester bonds
What are the major differences between RNA and DNA?
RNA is single stranded———–DNA double stranded
RNA has ribonucleotides———DNA deoxyribonucleotides
RNA has Uracil——————–DNA Thymine
RNA can fold into complex 3-D structures allowing some RNAs to have precise structural and catalytic functions
Ribose vs deoxyribose: which one has one less OH and is replaced with H?
Deoxyribose
Uracil vs Thymine: Which one has a methyl group instead of C-H?
Thymine
What direction is mRNA read?
5’->3’
What are mRNAs?
Messenger RNA that direct protein synthesis. Coding RNAs
3-5% of total cellular RNA
What is the final product of many genes?
RNA
Non-coding RNA serve as what?
Enzymatic and structural components for many biological processes
rRNA:
Ribosomal RNA form the core(basic structure) of ribosome and catalyze protein synthesis
snRNA:
small nuclear RNA direct the splicing of pre-mRNA to for mRNA
tRNA:
Transfer RNA form adaptors that select and hold amino acids during protein synthesis
snoRNAs
Small nucleolar RNAs, help to process and chemically modify rRNAs
siRNA:
Small interfering RNA regulate eukaryotic gene expression by degrading select mRNA
miRNAs:
Regulate gene expression by blocking translation of specific mRNAs and cause their degradation
Bacterial RNA polymerase structure
Multi-subunit complex
RNA Pol
Holoenzyme a2bbsigma
What is present at the active site in RNA polymerase?
Mg2+
Where do ribonucleosides enter the RNA Pol?
Through the ribonucleoside triphosphate uptake channel
What is the function of RNA Pol?
Catalyzes the formation of phosphodiester bonds that link nucleotides
Has proof-reading nuclease activity
Why can lower fidelity of RNA synthesis be tolerated by organisms?
Because mistakes are not transmitted to progeny
What are the general features of transcription?
- polymerase binds to promoter on DNA
- Unwinding of a portion of DNA
- Other strand acts as a template
- Complementary base pairing between nucleotides and DNA template
- Nucleotides covalently linked by phosphodiester bonds
- RNA sequence elongated 5’-3’ direction
- Released from DNA as single strand
What is the end result of transcription?
RNA sequence exactly complementary to the template strand and identical to the coding strand
What is the most important step in gene expression in prokaryotes? Why?
Initiation.
It is the main regulatory step; decides which proteins produced and at what rate
What are the steps of PT-initiation?
- RNA Pol assembles into holoenzyme and slides down the DNA until it locates a promoter
- The sigma subunit binds to the promotor and forms an open promoter complex
- Makes specific contacts with exposed bases
- Unwinds a short (17bp) segment of double-stranded DNA to form a transcription bubble
- One strand becomes template strand
- Transcription begins-short RNA formed via scrunching
- Polymerase still on promoter and pulls DNA to active site
- Stress causes abortive initiation with short RNA
What creates an open promoter complex?
Sigma subunit binding to promoter
What is a transcription bubble?
Short segment of double stranded DNA unwound by RNA Pol
What drives the reaction forward during PT transcription? What bonds are formed?
Hydrolysis of nucleoside triphosphates (ATP, CTP, UTP, and GTP) provides energy to form phosphodiester bonds and drive reaction forward.
What are the nucleoside triphosphates used in PT transcription?
ATP, CTP, GTP, UTP
What are promoters?
Special sequences of nucleotides that direct the RNA polymerase to the proper initiation site for transcription
What are the two promoter regions in prokaryotes?
There are two common motifs that are present on 5’ upstream side of the transcription start site
- 35 sequence (TTGACA)
- 10 sequence(TATAAT)
When does elongation in prokaryotes begin?
After the formation of nascent RNA with approximately to nucleosides
What are the steps of elongation in prokaryotes?
- RNA Pol breaks its interaction with the promoter
- the sigma factor dissociates from the DNA-RNA complex
- Core enzyme binds more strongly to DNA template
- RNA Pol moves stepwise, unwinding the helix just ahead of active site and exposing a new region for cbp
- Transcription continues till RNA Pol meets termination signals
When is transcription very efficient in prokaryotes?
During elongation
What does elongation generate in prokaryotes?
Superhelical tension
What eases superhelical tension in prokaryotes during elongation?
DNA gyrase
When does transcription end in prokaryotes?
When RNA Pol encounters termination signals on the DNA templates
What is the core enzyme of RNA Pol?
alpha2 beta betaprime
Sigma later adds on
Why is error tolerated in transcription?
Because mRNA/proteins only last for maybe hours or days but gets replaced
Which strand has a promoter?
The coding strand
What are the termination signals in prokaryotes during transcription?
When a string of AT nucleotide pairs preceded by a 2-fold symmetric DNA sequence
The RNA transcript of this region can form a self complementary hairpin structure followed by a poly (U) tail. AT transcribes into GC and the GC fold and bind to each other with a triple bond. This bond is very strong causing interaction of RNA with RNA Pol to destabilize.
In prokaryotes, all RNA molecules are synthesized by how many RNA Pol?
1
In eukaryotes how many RNA Pols synthesize RNA molecules?
3
Which RNA Pol transcribes all protein-coding genes, plus sno, mi, si, Inc, and most snRNA genes?
RNA Pol II
What is also required in eukaryotic transcription in addition to RNA Pol II?
General transcription factors
Why are general transcription factors needed for eukaryotic transcription?
Eukaryotic transcription needs to deal with higher order packing of DNA (nucleosomes, chromatin etc)
What are the unique features of RNA Pol II?
RNA Pol II contains a unique carboxyl-terminal domain of the 22-kD subunit called CTD
The activity of RNA Pol II is regulated by phosphorylation mainly on Ser residues of the CTD
Where do TFII assemble before transcription in eukaryotes?
at the promoter
What are the functions of TFII?
- to help position the RNA Pol correctly at the promoter
- aid in pulling apart the two strands of DNA to allow transcription to start
- release RNA pol from the promoter into the elongation mode once transcription has begun
What are the steps of Initiation in eukaryotic transcription?
- TFIID binds to TATA box (causes distortion in DNA)
- TFIIB is recruited
- TFIIE, TFIIF, TFIIH and RNA Pol II are recruited
- All these factors combine to form the transcription initiation complex
- TFIIH unwinds the DNA and exposes template
- RNA Pol II makes short lengths of RNA
- Phosphorylation of CTD by TFIIH causes the polymerase to leave the promoter and begin elongation
What subunit of TFIID recognizes TATA ?
TATA-box-binding-protein (TBP)
What is TFIIB purpose?
Accurately positions RNA Pol at the start site
What is the purpose of TFIIF?
Stabilizes RNA Pol interaction with TBP and TFIIB; attracts TFIIE, and TFIIH
What is the purpose of TFIIE?
Attracts and regulates TFIIH
What is the purpose of TFIIH?
Unwinds DNA at start point, phosphorylates Ser on CTD; release Pol from the promoter
What is the most common promoter region in eukaryotes?
TATA box
What are other additional regulatory sequences (promoters) for eukaryotic RNA Pol II?
CAAT and GC box
What happens during transcription elongation in eukaryotes?
RNA Pol II moves along the DNA transcribing RNA along the way
Elongation factors prevent dissociation of RNA Pol II until it reaches the termination signals
Superhelical tension
What are elongation factors?
Factors that prevent dissociation of RNA Pol II until itt reaches the term signals
What removes superhelical tension during transcription elongation in eukaryotes?
DNA topoisomerase
in vivo, describe hurdles that arise during eukaryotic transcription?
Since DNA is packaged in nucleosomes, which are arranged in higher order chromatin structures-these physical barriers prevent access to DNA
What proteins helps to attract RAN Pol II to transcription initiation start site?
Transcriptional activator
What protein complex allows the activator proteins to communicate with RNA Pol II and the general transcription factors?
The mediator
What are recruited in eukaryotic cells to provide greater access to DNA for transcription?
Chromatin remodeling complexes
Histone modifying enzymes
What is the immediate product of RNA Pol II?
pre-mRNA or primary transcript
What needs to happen to pre-mRNA before it is exported from the nucleus to cytosol for translation?
Needs to be processed and undergo covalent modifications to assess whether both ends of an mRNA molecule are present and message is intact
What three modification does pre-mRNA must undergo?
RNA capping: modification of the 5’ end
RNA splicing: removal of non-coding sequences
Polyadenylation: modification of the 3’ end
What does the Cap structure at the 5’ end consist of?
7-methylguanosine joined to 5’ end of RNA via a 5’-5’ triphosphate bridge
What is the role of the 5’ cap?
- helps to distinguish mRNA from other RNAs
- Defines the translational start site
- Stabilizes mRNA by protecting their 5’ ends from phosphatases and nucleases
- Binds a protein complex CBC (cap binding complex)
What is CBC?
Cap binding complex that helps in RNA processing and export
Why are eukaryotic genes discontinuous?
Because they contain exons and introns
Splicing:
Process when introns must be excised and exons linked to form final mRNA
What is splicing carried out by?
Spliceosomes - assemblies of proteins and small nuclear RNA
Where is the signal for polyadenylation
It is transcribed into mRNA
What are the unique consensus sequences for polyadenylation?
AAUAAA, GU or U rich sequences and CA
What recognizes the unique consensus sequences for polyadenylation?
RNA binding proteins and RNA processing enzymes
Important ones are
Cleavage and polyadenylation specificity factor (CPSF) and Cleavage stimulation factor (CstF)
What do the CPSF and CstF proteins travel on? Where are they transferred?
Both proteins travel on RNA Pol tail and are transferred to RNA as it emerges from the polymerase
What are the steps of 3’-polyadenylation?
- The AAUAAA at 3’ end of RNA is bound by CPSF
- The GU rich element beyond the cleavage site is bound by CstF
- CA sequence is 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
- Poly A binding proteins assemble
What is the source of A in polyadenylation?
ATP
What template is needed for PAP?
No template is required
mRNA that codes for only one protein?
monocistronic - most eukaryotes
mRNA that codes for multiple protein?
polycistronic - bacterial
What ends of bacterial mRNA are unmodified?
3’ and 5’ ends
Bacterial mRNA do not have _______. While their counterparts do and are removed.
introns.
**counterparts are eukaryotic cells
What does RNA Pol I make?
rRNA
What does RNA Pol II make?
mRNA precursor
What does RNA Pol III make?
tRNA
