8.11.16 Lecture Flashcards
Why do we use an RNA intermediate rather than have DNA transcribed directly to protein?
Utilizing an RNA intermediate allows for the expression of genes with different efficiencies. Many copies of RNA can be transcribed, leading to many proteins, OR a few copies can be transcribed, leading to only a few proteins.
What are the primary differences between RNA and DNA?
- RNA is a ribose; DNA is a deoxyribose.
- RNA uses U instead of T; DNA uses T instead of U.
- RNA is single stranded and folds into complex structures that can have catalytic activity; DNA is double stranded and forms a double helix.
Why does DNA use T instead of U (T is methylated U)?
T protects against nucleases, maintaining genomic integrity. In addition, C can spontaneously deaminate to U. If U were used normally, there would be no way to correct this mistake.
What are the 5 main types of RNA?
mRNA, rRNA, tRNA, snRNA, snoRNA
What does mRNA do?
Messenger RNA; code for proteins
What does rRNA do?
Ribosomal RNA; forms the basic structure of the ribosome and catalyzes protein synthesis
What does tRNA do?
Transfer RNA; translates RNA into proteins
What does snRNA do?
Small nuclear RNA; functions in a variety of nuclear processing, including splicing pre-mRNA
What does snoRNA do?
Small nucleolar RNAs; help process and chemically modify rRNA
RNA is synthesized in the ___ direction by addition of nucleoside monophosphates to the 3’-OH end of the growing RNA chain.
5’ to 3’
What are the steps of transcription?
- Initiation
- Elongation
- Termination
Describe the chain elongation reaction.
- An incoming ribonucleoside triphosphate is selected if it can base pair with the exposed template strand.
- A ribonucleoside monophosphate is added to the 3’-OH end o the RNA chain.
- The terminal 2 phosphate groups are released as pyrophosphate.
What catalyzes the chain elongation reaction?
RNA polymerase
What are the differences between transcription and DNA replication?
- Transcription uses ribonucleotides; DNA replication uses deoxyribonucleotides.
- Transcription uses RNA polymerase; DNA replication uses DNA polymerase.
- Transcription does not need a primer; DNA replication require a primer.
- Transcription is less accurate; DNA replication is more accurate.
- Transcription only copies pieces of the genome; DNA replication copies everything.
- Transcription makes many copies; DNA replication makes only one copy.
How many RNA polymerases do eukaryotes have? Prokaryotes?
3; 1; Note that mitochondria has its own polymerase
What is the location, products, and promoters for RNA polymerase I?
Location: Nucleolus
Product: pre-rRNA (5.8S, 18S, 28S rRNA genes)
Promoter: Ribosomal initiator element, upstream promoter element (UPE)
What is the location, products, and promoters for RNA polymerase II?
Location: Nucleoplasm
Product: Pre-mRNA, snoRNA, miRNA, siRNA, most snRNA
Promoter: TATA, CAAT, BRE, DPE, and others
What is the location, products, and promoters for RNA polymerase III?
Location: Nucleoplasm
Product: pre-tRNA, 5S rRNA, some snRNAs, other small RNAs
Promoter: A-box/C-box, A-box/B-box
What are the elements of a typical RNA polymerase II gene?
- Promoter region
- Transcription start site
- Translation initiation codon
- Exons
- Introns
- Translation termination codon
- 3’ end cleavage and polyadenylation signal
- Polyadenylation site
- Transcription termination site
What are the elements of a typical mRNA transcript?
- 5’ cap
- Translation initiation codon
- Translation termination codon
Where are promoters typically located?
Close to the start point, though can be upstream, downstream, or at the transcription start point.
True or False: several promoters work together to promote transcription.
True
True or False: the TATA box is essential for transcription by RNA polymerase II.
False
What do promoters do?
Mark the location and orientation of the gene to be transcribed
What determines which DNA strand serves as a template?
The orientation of the polymerase
Polymerase reads DNA in what direction? DNA is transcribed in what direction?
3’ to 5’
5’ to 3’
True or false: Promoters are asymmetrical.
True
What is the consensus sequence and general transcription factor for BRE?
G/C G/C G/A C G C C
TFIIB
What is the consensus sequence and general transcription factor for TATA?
T A T A A/T A A/T
TBP (subunit of TFIID)
What is the consensus sequence and general transcription factor for INR?
C/T C/T A N T/A C/T C/T
TFIID
What is the consensus sequence and general transcription factor for DPE?
A/G G A/T C G T G
TFIID
What are the three general requirements for a TATA box?
- T at the 3rd residue 100% of the time
- A at 6th residue 97% of the time
- AA points in the correct direction
___ are required for transcription initiation in eukaryotic cells.
General transcription factors
What are the general transcription factors for RNA polymerase II?
TFII
Describe the process of transcription initiation for RNA polymerase II.
- TFIID (through TATA Binding Protein [TBP]) recognizes and binds the TATA box. TFIID is comprised of TBP and numerous TATA binding protein associated factors (TAFs).
- TFIID distorts the DNA and marks the location of an active promoter.
- The rest of the general transcription factors (F, E, H) and RNA polymerase assemble at the promoter.
- TFIIH uses energy from ATP hydrolysis to pry apart the DNA double helix at the start point. It also phosphorylates RNA polymerase II, changing its conformation so that it is released from the general factors and can begin elongation. Phosphorylation occurs at the C-terminal domain (CTD).
True or False: without a TATA box, TBP cannot interact with the DNA.
False; TAFs can associate with the DNA in a sequence specific manner and force TBP to interact.
What is TFIIH?
Helicase
How does elongation occur?
RNA polymerase moves stepwise along the DNA, unwinding the helix at its active site. The polymerase adds nucleotides one by one.
The RNA transcript is a ___ copy of one of the two DNA strands.
Complementary
True or false: only one polymerase can work at a time.
False; multiple polymerases can work at the same time.
Describe transcription termination for the three different RNA polymerases.
- RNA polymerase I requires a polymerase specific termination factor that binds to the DNA downstream of the transcription unit.
- RNA polymerase II can terminate at multiple sites located over a distance of 0.5-2 kb beyond the poly (A) addition site.
- RNA polymerase III terminates after a series of U residues.
What must happen to the mRNA before translation?
The two ends are modified (5’ capping, 3’ polyadenylation), the introns are spliced out, and the mRNA is transported to the cytoplasm.
As RNA polymerase II transcribes DNA into RNA…
…it carries RNA-processing proteins on its tail that are transferred to the nascent RNA at the right time.
Describe the structure of the RNA polymerase II tail.
The tail contains 52 tandem repeats of a 7 amino acid sequence. There are two serines at each repeat. The capping proteins bind to the tail when it is phosphorylated on NSer5. The tail is then phosphorylated at Ser2 by a kinase, and eventually dephosphorylated at Ser5. These modifications attract splicing and 3’-end processing proteins.
True or false: the RNA polymerase II must be fully dephosphorylated to begin RNA synthesis as a promoter.
True
What purpose do the 7-methylguanine cap (5’ cap) and the polyA 3’ tail serve?
Identifies it as mRNA, helps export it, protects it from degradation, and helps maintain mRNA stability prior to translation.
What is unique about the 7-methylguanine cap?
It has a 5’-to-5’ triphosphate bridge.
Describe the process of capping an RNA molecule.
- Phosphatase removes a P.
- Guanylyl transferase adds GTP (releasing pyrophosphate)
- Methyl transferase adds a methyl group.
- Sometimes an additional methyl group is added.
How is mRNA synthesis terminated in eukaryotes?
By cleavage of the pre-mRNA from RNA polymerase II while it is still synthesizing RNA.
How is mRNA synthesis terminated in prokaryotes?
RNA polymerase stops at a termination signal and releases the transcript and DNA template.
What directs the cleavage and polyadenylation of the 3’ end of a eukaryotic mRNA?
RNA sequences encoded in the genome are recognized by specific proteins
Describe the process of polyadenylation and termination.
- When the signals are reached, the hexamer AAUAAA is bound by CPSF (cleavage and polyadenylation specificity factor) and the GU-rich element is bound by CstF (cleavage stimulation factor). The CA sequence is bound by a third protein factor.
- Poly-A polymerase (PAP) leads to the cleavage of RNA; poly-A-binding proteins bind; RNA polymerase eventually terminates.
- Additional poly-A-biding protein is added.
How is an intron defined?
- SR proteins mark the borders (border proteins)
- GU always comes directly after the 5’ splice site (splice donor).
- A is always the branch point, which occurs 20-50 bp upstream from the splice acceptor.
- AG always occurs just before the 3’ splice site (splice acceptor)
Splicing involves what reaction? What happens in this reaction?
2 transesterification reactions; -OH on the branch-point A attacks the P of the first exon, splicing this end. The OH of this exon attacks the P of the second exon. An excised lariat intron and spliced exon is formed.
What catalyzes the splicing reaction?
A small nuclear RNA-protein complex (snRNP) called the spliceosome (a ribonucleoprotein complex)
Describe the canonical splicing reaction.
- U1 snRNP base pairs with the 5’ splice junction and the BBP (Branch-point binding protein) and U2AF (U2 auxiliary factor) recognize the branch-point site.
- U2 displaces BBP and U2AF and forms base pairs with the branch-point site consensus sequence.
- The U4/U6-U5 “triple” snRNP enters the reaction. U4 and U6 are firmly bound. Subsequent rearrangements break these apart, allow U6 to displace U1 at the 59 splice junction. This creates the active site that catalyzes the first phosphoryl-transferase reaction, splicing at the 5’ site. Note the formation of the intron lariat.
- Additional RNA-RNA rearrangements creative the active site for the second reaction, which completes the splice.
- The two exon sequences are joined by the exon junction complex (EJC).
How do snRNPs guide splicing?
By base-pairing with complementary sequences in the mRNA transcript.
What happens in beta-thalassemia (decreased production of Hb)?
G to A mutation inactivates a normal splice site at the end of exon 1 in the beta-globin gene, leading to activation of three adjacent cryptic 5’ splice sites. C to G mutation in intron 2 creates a new 5’ splice site and activates an adjacent cryptic 3’ splice site. A new exon is incorporated.
How is mRNA exported after processing?
A variety of proteins bind to the new mRNA in the nucleus during and after processing, including proteins that bind to the exon-exon junctions, cap-binding proteins, and polyA binding proteins. Some remain bound as it is exported; others are removed. SR proteins help protect the mRNA and get it out of the nucleus.
Describe transcription of an rRNA gene by RNA polymerase I.
- Upstream binding factor (UBF)
- TATA binding protein (TBP) - a universal transcription factor required by all three RNA polymerases
- Note there is no TATA box in the promoter, but TBP interacts in a sequence independent manner.
- SL1 binds to UBF
rRNA genes are present in multiple copies. Humans contain ___ rRNA genes per haploid genome.
200
What is the site of rRNA synthesis and packaging?
Nucleolus
What are the 3 rRNAs synthesized by RNA polymerase I?
18S, 5.8S, 28S (synthesized as a large 45S precursor)
What are the two types of chemical modifications on rRNAs? What performs these modifications?
- Methylation of the 2’-OH position on nucleotide sugars
- Isomerization of uridine to pseudourine
snoRNAs complexed with snoRNPs that contain the guide sequences and the enzymes to modify the RNA.
What is synthesized by RNA polymerase III?
5S (fourth rRNA) and tRNA
Describe the components of the pre-5S rRNA gene.
- TBP
- Pol III
- TFIIIA, binds to the C-box
- TFIIIB
- TFIIIC
Describe the components of the pre-tRNA gene.
- TBP
- TFIIIB, binds to B-box
- Pol III
What types of modifications can occur on pre-tRNA?
- Removal of a short intron in the anticodon loop
- Removal of a short nucleotide sequence from the 5’ end of the pre-tRNA
- Replacement of 2 U at the 3’ end with CCA
- Modification of specific bases in the tRNA
___ bases will be modified in tRNA. Why?
1/10; critical for folding
