RNA vs. DNA
- RNA has 2’ hydroxyl. DNA does not.
- RNA is more transient because 2’ hydroxyl can do nucleophilic attach on the phosphate and degrade itself.
- DNA is templated off of DNA to a double strand. RNA is produced in a single stranded way.
- RNA has more structural flexibility. can adopt more confirmations.
- RNA can also have enzymatic functions
What are the 3 classes of RNA and some examples?
- structural: rRNA, tRNA (transfer), snRNA, snoRNA (small nucleolar)
- regulatory: miRNA (naturally occuring), siRNA (small interfering, most of these are generated for bio research tools.
- information containing: mRNA
defects in aminoacyl tRNA synthetase genes and proteins can often cause what kind of diseases?
RNA’s role in ribosomes
ribosomes have two subunits and MOST of this structure is the RNA, and the enzymatic activity of the ribosome is mainly carried out by the RNA
DNA directed RNA synthesis, first step to extract information unidirectionally from DNA
why is transcription unidirectional?
RNA synthesis can only go in one direction, RNA polymerase is processive and can only proceed in one direction.
What does processive mean with relation to RNA pol?
Once an RNA pol starts txn, it has to finish! It can’t just leave off and let someone else pick up the slack.
what is the product of transciption
primary transcript: a copy of all the information in the transcription unit
what is a txn unit?
the ORF, UTRs, and the introns
what does a promotor
directs RNA pol to the start of a gene. it controls directionality and frequency of txn.
What is the definition of a gene? from a molecular biology standpoint
Txn unit and the flanking regulatory sequences
What is the TATA box?
it is a common sequence in pomoters. the sequence is TATAAA
examples of inherited diseases caused by mutations in Txn regulatory genes
sex reversal (SRY), dwarfism (PIT-1), X linked mental retardation ATR-X syndrome, (XH-2)
(side note) txn factors are also a common drug target (tamoxifen, cyclosporinA))
what is transcriptome profiling?
it is a powerful diagnostic tool where you sequence mRNA from a cell (transcriptome). allows design of the most effective theraputic response is for a particular class of tumor.
formation of RNA chain
- nucleophilic attach of 3’ OH onto the alpha phosphate on incoming nucleotide. releases pyrophosphate. which then itself becomes hydrolyzed. this energy is used to move RNA pol along the DNA template.
RNA template relative to the template strand and the nontemplate or coding strand.
RNA is complementary to the template strand and is identical (almost) to the coding strand.
function of RNA polymerase I
synthesis of rRNA (the busiest one!)
function of RNA polymerase II
synthesis of mRNA, snRNA, miRNA, lncRNA
Function of RNA polymerase III
synthesis of tRNA, 5S RNA, U6snRNA, 7SK RNA
what is the Xist RNA?
a lnc, long non coding RNA that is required for x inactivation
Steps of Txn (3)
- Initiation (also step 1-3)
- elongation (also step 4)
- termination (also sept 5)
Steps of initiation of txn.
- RNA pol binds to promotor in closed complex
- polymerase melts duplex to make open complex with txn bubble
- first two NTPs come in and RNA pol makes the first phosphodiester linkage between the two
Description of elongation
promoter espcape leads to movement of RNA pol 3’ to 5’ down the DNA template. This is not a smooth continous movement. it pauses, starts, which may be important regulatory steps in controlling txn. it proceeds, melting DNA and adding NTPs. assisted by sliding clamp, to keep RNA pol attached to DNA, but still allowing RNA pol to move along it.
two key steps that a subject to regulation of RNA transcript in txn
2. elongation, especially related to restarting a paused RNA pol
what is so special about CTD region on RNA pol II specifically (the others dont have this)
this area has a reversibly phosphorylated heptad repeats in the C-terminal domain, (CTD) which binds to proteins that function in elongation and processing of the RNA transcript (such as splicing factors)
Tell me about alpha amanitin
come from death cap mushroom. It forms a tight bond with the bridge helix that connects the two RNA subunits. It prevents translocation of the RNA pol along the DNA
Tell me about the channels of RNA pol II
There is one where the DNA is fed into the enzyme, Where DNA is fed out, Where RNA exits, and a secondary channel where NTP substrates diffuse into the active site
What is one peptide that is shared among the general Txn factors for RNA Pol I, II, and III? And why is it so special, regarding its binding?
TBP, tata binding protein, is found in general txn factors that work with RNA pol I, II, and III. These factors assist RNA pol in recognizing the promoter (the enzyme cannot do this alone). TBP binds the TATA sequence and binds DNA in the Minor Groove! because of this it is a relatively non sequence specific binding protein
TFIID: what is it and what does it recognize?
A complex of proteins containing TBP subunit, Recognizes sequences in the promoter, (is a promoter recognition factor) specifically recognizes TATAAA sequence, 30 bp upstream of start site
TFIIH (what does it do, why is it notable?)
- CDK7 protein kinase phosphorylates the CTD heptad repeats of RNA pol II during promoter release
- XPB helicase pulls apart DNA at promoter to transition from closed complex to open complex
- DNA repair!
TFIIH mutation can lead to which diseases?
Xeraderma pigmentosa (DNA repair problem, lower level of unscheduled DNA synthesis (UDS), ie. repair), Cockayne’s syndrome, trichothiodystrophy (TTD) (both of these are associated with txn problems. TTD also has lower UDS)
What are the components of the pre-initiation complex?
RNA pol II holoenzyme, DNA binding txn factor, Co-activators, moderator
How is this pre initiation complex formed?
Sequence specific DNA binding TF (like P53), and the protein binding domain recruits other general TFs (TFIID), and with coactivators (many of these modify chromatin structure), and mediator, which mediates signals from TFs.
What are the 3 main mRNA processing steps?
5’ capping (first), splicing of introns, and maturation of 3’ end
describe 5’ capping
- Gamma (terminal) phosphate is removed, by triphosphatase,
- GMP is added in a 5’-5’ linkage (backwards) to the first mRNA residue (at the 5’ end), with 3 phosphates in the middle. by guanylyl trnasferase
- The guanisine is then methylated at 7’ position, consuming S adenosylmethionine (which then become s adenosylhomocysteine) by guanine 7 methyl transferase.
What is the universal methyl donor?
purposes of the 5’ cap
- protects 5’ end from exonucleases
- site of binding for CBC cap binding complex in nucleus
- site of binding of Eukaryotic initiation factor 4E, EIF4E in cytoplasm
- When it is removed in the cytoplasm it stimulates degradation.
What are the functions of CBC (cap binding protein)
This is a heterodimer that 1. enhances subsequent processing steps and 2. enhances export of mRNA to cytoplasm
What are the functions of EIF4E, (Eukaryotic initiation factor 4E)
stimulates translation on ribosomes
What happens when you overexpress EIF4E?
It triggers malignant transformation, it deregulates translation.
What is the 5’ conserved sequence for splicing, in the introns
GT (in DNA) or GU (in RNA)
What is the branch point for splicing in the Intron, and why is it special?
A, its the residue that initiates the first step of the splicing process.
What is the 3’ conserved sequence for splicing, in the introns
Which components of the splicosome recognize which splicing specific sequences in the intron?
GU at 5’ end by the U1 snRNA, A at branch point, U2 snRNA, AG at 3’ end by the U2 associated factor, U2AF.
What are splicing enhancers?
RNA sequence elements that can be present in exons or introns and enhance splicing
What are splicing silencers?
RNA sequence elements that can be present in exons or introns and inhibit splicing
How does the splisosome differentiate between exons and introns? (Which one does it define?)
Exon definition: formation of a complex between U1, U2, U2AF, and other proteins that bind the exon. This forms the cross exon recognition complex.
side note: splisosome is comprised of about 5 snRNA and over 100 proteins.
Describe the splicing process
It is accomplished by 2 transesterification reactions, so the number of phosphodiester bonds is always constant.
- Branch point A becomes squeezed out after binding to U2. the 2’ hydroxyl (OH) carries out nucleophilic attack on the phosphodiester bond at junction of exon and intron. Cleaves that bond. Liberate 5’ exon, expose 3’ OH
- That 3’ OH, at end of the upstream exon does nucleophilic attach on phosphodiester bond between intron and downstream exon. This joins exon 1 and 2. intron is released as a lariat structure, where the branch point is making 3 phosphodiester bonds, at 2’, 3’, and 5’ hydroxyls.
What is alternative splicing? Can it go wrong?
A single primary transcript can be spliced in multiple ways. greatly enhances coding potential of human genome.
- exon skipping
- mutually exclusive exons,
- alternative 3’ splice sites, to extend 5’ end of an exon
- alternative 5’ splice sites, to extend 3’ end of an exon
- retention of intron.
Regulation of splicing is commonly disrupted in human diseases
describe maturation of 3’ end
- endonucleolytic cutting of RNA, 20 bp downstream from highly conserved AAUAAA in RNA, (AATAAA in DNA), exposing 3’ OH,
- polyadenylation at that 3’ OH, by polyadenylate polymerase, 150-200A
What is the function of the poly A tail?
- binds to poly A binding protein, stabilizing RNA, protection from 3’ end degradation
- enhanced translation.
- along with cleavage, polyadenylation is coupled to termination of txn. (so if you mutate a polyA site, you do not get proper termination of txn)
Significance of alternative Polyadenylation, for example with regards to IgGs
This occurs on more than have of mRNAs. For the IgG heavy chain, alternative Polyadenylation can affect whether it is a lymphocyte membrane bound or serous protein.
What is the initiation codon, and what does it code for?
AUG, for methionine
What are the termination codons? Does it/they code for an amino acid?
UAA, UAG, UGA, These do not code for an amino acid.