What are 3 differences between RNA & DNA?
- 1. the pentose sugar in RNA is ribose instead of 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
- the sequence of DNA that is copied during the synthesis of mRNA
- (RNA is synthesized 5' → 3’)
- the strand with a base sequence directly corresponding to the mRNA sequence
- this sequence corresponds to the codons that are translated into protein
- also known as the mRNA-like strand, or (+) strand
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
What are the 3 eukaryotic polymerases involved in RNA synthesis?
- • RNA polymerase I, II & III
- • they transcribe different genes
- • are complexes made up of protein subunits
- • while some subunits are found in all 3, they also have nonidentical subunits, which explains why they’re responsible for DIFFERENT jobs
RNA polymerase I
• found in the nucleOlus & transcribes 28S, 18S & 5.8S rRNAs (ribosomal RNAs)
• is important for PROTEIN SYNTHESIS
RNA polymerase II
• found in the nucleus & transcribes pre-mRNA (heterogeneous nuclear RNA)
• is important for protein encoding, RNA splicing, & post-transcriptional gene control
• is incapable of locating a promoter in the absence of the basal transcription factors
RNA polymerase III
• found in the nucleus and transcribes tRNA, 5S rRNA & other small RNAs
• small RNAs are involved in the splicing process
How can different polymerases be distinguished?
by their sensitivities to α-amanitin
- pol I: resistant
- pol II: sensitive
- pol III: intermediate
What appears to be the evolutionary origin of eukaryotic RNA polymerases?
because there are so many identical subunits, it appears that many of the eukaryotic RNA polymerase components arose via gene duplication & subsequent sequence divergence
Pol I rRNA Synthesis
1. rRNA transcription units are arranged in multiple, tandemly arrayed copies in the genome; individual units are separated by a spacer
2. the rRNA transcription unit is transcribed as a SINGLE RNA chain that’s 13,700 nts long
3. 2 transcription factors (B & S) promote binding of RNA Pol I to the “regulatory region”
What are the transcription factor requirements for rRNA synthesis by pol I?
• factor B (for binding) binds to sequences within the regulatory region
• its binding is greatly stimulated by a 2nd factor called the S factor
• upon assembly of these 2 factors, RNA pol I can bind & transcription begins
• (transcription terminates past the 28S rRNA near the next start site in the array)
What marks the termination site of RNA pol I?
a string of T residues
Which two transcription factors are required for RNA pol I to bind to & transcribe DNA?
transcription factors B & S (B for binding, S for stimulating)
What purpose do rRNAs that complex with ribosomes serve?
they help build ribosomes (structural) & have catalytic activity
What are the two subunits of eukaryotic ribosomes?
60S & 40S
the 28S & 5.8S rRNA associate with the 60S ribosomal subunit
the 18S associates with the 40S ribosomal subunit
What RNA pol transcribes the many small RNAs found in eukaryotic cells?
RNA Polymerase III [eg. tRNAs, RNAs that assemble in small nuclear ribonucleoproteins (snRNPs), 5S rRNA & many others whose functions are not known]
Synthesis of 5S rRNA (by pol III)
• made up of 120 nucleotides & is a component of the large ribosomal subunit
• the activation region for 5S rRNA lies WITHIN the gene
• assembly factors TFIIIA & TFIIIC bind to this activation region & enable the binding of TFIIIB (a transcription factor)
• RNA pol III recognizes TFIIIB (+ other factors) → transcription begins
• termination occurs at a string of T residues
• the primary transcript is processed by removal of several bases from the 3′ end (other Pol III transcripts, eg. tRNA, are more extensively processed)
Where are promoters for transcription of RNA via Pol III located?
Within the genes!
What transcription factors are required to recruit & activate RNA Pol III?
TFIIIA, TFIIIB & TFIIIC (the Roman numeral in the name of the tx factor tells you which pol it’s 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 where RNA polymerase binds to initiate transcription
crucial to the accuracy & rate of transcription initiation
are usually located immediately upstream of the gene
Upstream v. Downstream
upstream: OPPOSITE to the direction of transcription
downstream: in the SAME direction as transcription
- sequences of DNA that can up-regulate transcription of neighboring genes
- - they exert their effects over long distances, function in either orientation & are located upstream, downstream or in a given gene
What is common about all genes’ regulatory regions?
the TATA box - directs RNA pol II binding, found ~25 bases upstream of the gene (-25)
is HIGHLY conserved
What happens at the TATA box?
basal transcription factors assemble
specifically, TATA-binding protein (TBP), a subunit of TFIID, binds
Basal Transcription Factors
transcription factors required for the initiation of transcription (DNA → RNA) in all genes
• in response to injunctions from activators, these factors position RNA pol II at the start of the protein-coding region of a gene
• RNA pol II is incapable of locating a promoter without basal transcription factors
What is the only basal transcription factor that binds to DNA?
TFIID, a subunit of which is TBP (TATA-binding protein)
TFIID phosphorylates pol II, activating it
a basal transcription factor that interacts with TFIID in addition to binding to pol II & help direct it to the promoter
• sequences of DNA that can up-regulate transcription of neighboring genes
• can exert their effects over long distances & function in either orientation
• are located upstream, downstream or within a given gene
• TFs that bind to enhancers are called Activators
Where are pre-mRNA transcripts processed?
What are the 3 processing steps pre-mRNA goes through before it becomes mRNA?
1. 5' 7-Methylguanylate CAP
2. 3' poly-A tail [a feature of RNA pol II transcripts only]
3. intron splicing, exon ligation
During processing what is added to 5’ end of nascent RNA?
7-methylguanylate (m7G) cap
• it prevents the RNA from being degraded & later signals for translation to occur
• it’s attached via a 5' → 5' triphosphate bridge
What goes on the 3’ end of the nascent RNA?
a ~250 nt poly-A tail
• it’s not placed where the mRNA transcript has stopped being made, it’s added UPSTREAM of the end of the entire primary transcript
• there’s a cleavage event & then the poly A tail is added
What sequence signals for cleavage of pre-mRNA & addition of the poly(A) tail?
located 10-30 nucleotides upstream of the site of cleavage & subsequent polyadenylation
CPSF (Cleavage & Polyadenylation Specificity Factor)
recognizes the AAUAAA sequence on pre-mRNA & recruits poly(A) polymerase
• upon cleavage, poly(A) polymerase adds ~250 adenylate residues
a polymerase that adds the poly-A tail without using a template
• poly-A tails are NOT encoded within genes
Which comes first, the 5’ or 3’ pre-mRNA processing?
the 5’ cap, THEN the polyA tail is added
part of primary transcript but not found in mature mRNA
• variable both in number (eg. 2 in β-globin & 50 in collagen) & length (up to ~200 kb)
the part of a eukaryotic gene coding for RNA which will ultimately be spliced into functional mRNA
• are thought to contain individual functional domains
the process by which introns are removed from the pre-mRNA molecule
a large ribonucleotide protein complex similar to the ribosome that contains smaller nuclear snRNPs (U1, U2, U4, U5 & U6) that aid in the removal of introns from pre-mRNA molecules
splicing takes place on the spliceosome
Pre-mRNA introns contain:
• GU residues at their 5' end
• AG residues at their 3' end
• a pyrimidine-rich region just upstream of the 3' splice site
What is the first step in splicing?
cleavage of the 5' exon-intron junction
Steps of Splicing
1. cleavage at the 5’ splice site → formation of a lariat
2. cleavage of the RNA at the 3’ splice site with concomitant joining of the two exons
3. the 3' intron-exon junction is cleaved & the neighboring exons are ligated together
the circularization of the intron with a Branch Point between the 5' G residue linked (5' to 2') to an Adenosine residue located near the 3' end of the intron
Match the snRNP with the exon-intron junction it binds:
U1 → 5' exon-intron junction
U2 → branch point (GU → A)
U5 → binds just upstream of the 3' intron-exon junction
(Several additional snRNPs are required to form a spliceosome)
• characterized by defective synthesis of 1 or more hemoglobin chains as a result of mutations that prevent proper intron splicing
• hereditary abnormalities of hemoglobin production in which there is a quantitative deficiency of either β-globin (β- thalassemia) or α-globin (α-thalassemia)
a single pre-mRNA can be spliced in different ways, yielding different mature mRNAs; a single gene can code for a series of related, but non-identical proteins
• accounts for much of our diversity
eg. alpha-tropomyosin (muscle contraction protein)
How is it that immunoglobulins of the IgM class exist either as membrane bound proteins displayed on the cell surface or as soluble proteins secreted into the blood?
soluble proteins lack two exons that encode very hydrophobic amino acids that normally anchor the membrane bound form on the cell surface
What are 2 inhibitors of transcription?
1. Rifampicin: blocks initiation of bacterial RNA synthesis (inhibits bacterial RNA pol)
2. Actinomycin D: binds to dsDNA & prevents it from being used as a template (inhibits mammalian RNA pol I)
Primary Transcript v. cDNA
• Primary Transcript: RNA that has both introns & exons
• cDNA: DNA copy of mRNA, therefore is missing introns & includes polyA tail + post-trascriptional modifications
cDNA Library Creation
• used to clone individual mRNA molecules
1. start with cells that express the gene of interest
2. isolate mRNA using oligo (dT) columns to selectively remove poly A containing mRNA
- 3. mix isolated mRNA with oligonucleotides containing ~20 deoxythymidines
- - these oligonucleotides act as primers for reverse transcriptase
- 4. reverse transcriptase catalyzes the synthesis of a DNA copy of the mRNA molecule
- - end up with an RNA-DNA hybrid
5. reverse transcriptase is over zealous & begins to copy the newly synthesized DNA strand
6. this material is used by a DNA pol to synthesize the second strand
7. hairpin loop is cleaved by S1 nuclease, a single strand specific nuclease
8. cDNA can be ligated into a suitable vector (either plasmid or phage)
- 9. cDNA containing vector is then introduced into bacterial cells
- - plasmid vectors frequently contain a gene that confers resistance to ampicillin; bacteria lacking the plasmid do not survive antibiotic selection
10. resulting collection of cloned cDNA fragments propagated in bacteria is called a cDNA library
True or False: Genes that are constitutively expressed contain a TATA box as a promoter?
False. They generally contain a GC-rich region instead.
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:
Which rRNAs make up the 40S subunit of eukaryotic ribosomes?
Which rRNAs make up the 60S subunit of eukaryotic ribosomes?
28S, 5.8S & 5S rRNAs
Which rRNA is synthesized outside of the nucleolus?
5S rRNA (it is synthesized by RNA poly III)