BioChem RNA Synth (4)
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List three differences between RNA and DNA:
- 1) the pentose sugar in RNA is ribose rather than deoxyribose
- 2) RNA has Uracil (U) instead of Thymine (T)
- 3) DNA normally exists as a double helix whereas RNA exists as a single strand.
True or False: mRNA typically exists as a single-stranded molecule
the sequence of DNA that is copied during the synthesis of mRNA
coding strand/mRNA-like strand
the strand with a base sequence directly corresponding to the mRNA sequence (the sequence corresponds to the codons that are translated into protein)
The sequence of RNA transcripts is similar to that of which DNA strand?
The coding strand (also called the (+) strand). The other strand is called the template or (-) strand.
In which direction is RNA synthesized?
5' to 3'
Name the three eukaryotic polymerases involved in RNA synthesis:
RNA polymerase I, II and III
What is the cellular location and function of RNA polymerase I?
It is found in the nucleolus and transcribes 28S, 18S and 5.8S ribosomal RNAs (rRNA); important for PROTEIN SYNTHESIS
What is the cellular location and function of RNA polymerase II?
It is found in the nucleus and transcribes pre-mRNA; important for PROTEIN ENCODING, RNA SPLICING, POST-TRANSCRIPTIONAL GENE CONTROL
What is the cellular location and function of RNA polymerase III?
It is found in the nucleus and transcribes tRNA, 5S rRNA and other small RNAs
What are the sensitivities of polymerase I, II and III to alpha-amanitin?
- pol I = resistant
- pol II = sensitive
- polIII= intermediate
most mRNA processing steps take place in the:
rRNAs that complex with ribosomes serve what purpose?
- 1) help build ribosome (structural)
- 2) catalytic activity
What are the two subunits of eukaryotic ribosomes?
- 60S and 40S
- The 28S and 5.8S rRNA associate with the 60S ribosomal subunit, and the 18S associate with the 40S ribosomal subunit.
Which two transcription factors are required for RNA polymerase I to bind to and transcribe DNA?
Transcription factors B and S (B for binding, S for stimulating)
What marks the termination site of RNA poly I?
A string of T residues
Where are promoters for transcription of RNA via Pol III located?
Within the genes!
What transcription factors are required to recruit and activate RNA Pol III?
- TFIIIA, TFIIIB and TFIIIC (note that the Roman numeral in the name of the transcription factor tells you which polymerase it is associated with)
- - TFIIIA & TFIIIC bind to the activation region located IN the gene
- - this recruits TFIIIB to bind which subsequently recruits RNA Pol III
- -termination occurs at a string of T residues
regions of DNA crucial to the accuracy and rate of transcription initiation; are usually located immediately upstream of the gene
What sequence in the promoter region directs Pol II binding? What protein binds this sequence?
The TATA box, it's HIGHLY conserved. The most common sequence is TATAAA. This is bound by the TATA-binding protein (TBP) a subunit of TFIID.
Basal Transcription Factors
transcription factors that are required for the initiation of transcription (DNA --> RNA) in all genes
What is the only basal transcription factor that binds to DNA?
TFIID, a subunit of which is TBP (TATA-binding protein)
a basal transcription factor that interacts with TFIID in addition to binding to pol II and help direct it to the promoter
RNA pol II is _________ of locating a promoter in the absence of the basal transcription factors
Enhancers ['volume control']
- sequences of DNA that can up-regulate transcription of neighboring genes
- -can exert their effects over long distances
- -can function in either orientation
- -are located upstream, downstream or within a given gene
- -TFs that bind are called activators
pre-mRNA transcripts are processed in which organelle?
What are the 3 processing steps pre-mRNA goes through before it becomes mRNA?
- 1) 5' 7 methylguanylate CAP
- 2) 3' poly-A tail
- 3) intron splicing, exon ligation
What goes on the 5 end of the nascent RNA?
A methylguanylate (m7G) cap; it prevents the RNA from being degraded and later signals for translation to occur
How is the m7G residue attached to the 5' end of an RNA?
via a 5' to 5' triphosphate bridge
What goes on the 3 end of the nascent RNA?
poly-A tail; added upstream of the end of the entire primary transcript
Which sequence element signals for cleavage of hnRNA and addition of the poly(A) tail?
The AAUAAA sequence located ~15 nucleotides upstream of the site of cleavage and subsequent polyadenylation.
True or False: Poly A tails are encoded within genes?
False. The tails are added by poly(A) polymerase, a polymerase that does not utilize a template. The tail is ~250 A's long.
What transcription factor reads the AAUAAA sequence on pre-mRNA?
Cleavage and polyadenylation specificity factor (CPSF); it recruits poly(A) polymerase and upon cleavage, poly(A) polymerase adds ~250 adenylate residues.
What is RNA splicing?
The process by which introns are removed from the pre-mRNA molecule
a large ribonucleotide protein complex akin to the ribosome that contains smaller nuclear snRNPs (U1, U2, U4, U5 and U6) which aid in the removal of introns from pre-mRNA molecules
Introns in pre-mRNA contain __ residues at their 5' end and __ residues at their 3' ends. Introns also contain a __________-rich region just upstream of the 3' splice site.
- GU residues at their 5' end
- AG residues at their 3' end
- Introns also contain a pyrimidine-rich region just upstream of the 3' splice site
The first step in splicing is:
cleavage of the 5' exon-intron junction
What is a lariat?
the circularization of the intron with a branch point between the 5' G residue linked (5' to 2') to an A residue located near the 3' end of the intron.
The last step in splicing is:
the 3' intron-exon junction is cleaved and the neighboring exons are ligated together
Match the snRNP with the exon-intron junction it binds:
- U1 binds to the 5' exon-intron junction
- U2 binds to the branch point (G --> A)
- U5 binds just upstream of the 3' intron-exon junction
alpha & beta thalassemias
characterized by defective synthesis of one or more hemoglobin chains as a result of mutations that prevent proper intron splicing
What is alternative splicing?
- A single pre-mRNA can be spliced in different ways, yielding different mature mRNAs. Thus, a single gene can code for a series of related, but non-identical proteins.
- eg. alpha-tropomyosin
Which rRNAs make up the 40S subunit of eukaryotic ribosomes?
18S rRNA (and proteins)
Which rRNAs make up the 60S subunit of eukaryotic ribosomes?
28S, 5.8S and 5S rRNAs (and proteins)
Which rRNA is synthesized outside of the nucleolus?
5S rRNA (it is synthesized by RNA poly III)
Name two inhibitors of transcription:
- 1) Rifampicin: blocks initiation of bacterial RNA synthesis
- 2) Actinomycin D: binds to ds-DNA & prevents it from being used as a template
Which RNA polymerase is inhibited by the drug actinomycin D?
Mammalian RNA polymerase I
Which RNA polymerase is inhibited by the drug alpha-amanitin?
Mammalian RNA polymerases II and III
Which RNA polymerase is inhibited by the drug rifampicin?
Bacterial RNA polymerase
True or False: The activation region for the 5S rRNA gene is found within the gene itself?
True or False: Genes that are constitutively expressed contain a TATA box as a promoter?
False. They generally contain a GC-rich region instead.
What is the only basal RNA polymerase II transcription factor that directly binds to DNA?
What is the function of the RNA Gppp cap?
It is believed to protect against the degradation of nascent RNA
True or False: All RNAs acquire a poly(A) tail following transcription?
FALSO, only those RNAs that are to become mRNAs acquire a poly(A) tail.
Introns in pre-mRNA typically have ___ residues at their 5' end and ___ residues at the 3' end:
What is the first step of RNA splicing?
Cleavage of the 5' exon-intron junction by the spliceosome
What is an intron lariat?
It is an intron that has been circularized with a specific branch point between a 5' G residue and a 3' splice-site A residue linked in a 5' to 2' bond.
Which small nuclear ribonuclearprotein (snRNP) binds to the 5' exon-intron junction?
Which small nuclear ribonuclearprotein (snRNP) binds to the branch point of an intron lariat?
Which small nuclear ribonuclearprotein (snRNP) binds upstream to the 3' intron-exon junction?
How is mRNA isolated for cDNA library creation?
oligo (dT) columns are used to selectively remove poly A containing mRNA
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