Exam 4
Hijacking the RNAi system
"Knockdowns" of protein-coding genes allows a pseudo-genetics strategy in human cells can be very specific you can make it target only one protein to eliminate
rRNAs are produced by RNA polymerases I & III
- 25S, 18S and 5.8S rRNAs are cleaved apart, modified and processed prior to and after export from the nucleus - 5S rRNA is made by Pol III and lightly modified
caps & viruses & defenses
- All cellular mRNAs are capped during transcription - Upon viral infection, infected cells turn off cap-dependent protein translation - Some viruses can also interfere with cap-dependent translation by decapping or killing eIF4E/G - Some viruses can also initiate cap-independent protein translation using structural tricks...
Kozak's Rules are a set of requirements
- Best context uses A of ACCAUGG as +1: ¤Purine in -3 position ¤G in +4 position -5-10% cases ribosomal subunits bypass 1st AUG scanning for more favorable one
snRNAs and snoRNAs
- Biogenesis starts out like mRNAs (by RNA Pol II) - After capping, they take a different turn - The cap helps direct them out to the cytoplasm
L1 elements in action
- Hemophilia caused by germ-line L1 movement Factor VIII gene was interrupted by a transposed L1 element - not an essential gene, but blood clotting disorders result from this.
Post-acylation editing of some aa-tRNAs
- In prokaryotes the initiator tRNAfMet is first charged with methionine. - Methionyl-tRNA formyltransferase then catalyzes formylation of the methionine, using tetrahydrofolate as formyl donor, to yield formylmethionyl-tRNAfMet - In some prokaryotes, a non-discriminating aaRS loads aspartate onto tRNAAsn. - The aspartate moiety is then converted by an amido-transferase to asparagine, yielding Asn-tRNAAsn. - Glu-tRNAGln is similarly formed and converted to Gln-tRNAGln in such organisms.
Decapitated translation initiation
- Many viruses encode RNAs that are uncapped - Upon shutting down cap-dependent translation, it focuses the ribosomal action on its own mRNAs - But you told us that caps are required for translation initiation in eukaryotes... - What's up? - Special structures called IRES elements (internal ribosome entry site) allow for mRNA recognition in the absence of cap - 40S ribosomal subunit binds directly to Hepatitis C virus IRES in the absence of most initiation factors - A dramatic change in the conformation of the 40S subunit occurs when it binds Hepatitis C virus IRES setting the AUG at the P site - Different viruses use different strategies for cap-independent translation. This is an example of the poliovirus strategy. ***Mechanism: Viral protease clips off N-terminus of eIF4G, so it can't bind eIF4E. eIF4G binds a viral protein (X), that binds to the IRES, promoting translation of the uncapped viral mRNAs.
64 combinations possible
- No organisms encodes all 64 combinations of tRNAs, although there are often more than 64 tRNA genes in the genome - How does nature ensure all 64 codons will be recognized? - tRNAs go wobbly... There are 61 codons specifying 20 amino acids. Minimally 31 tRNAs are required for translation, not counting the tRNA that codes for chain initiation. Mammalian cells produce more than 150 tRNAs.
What is RISC?
- RNA Induced Silencing Complex - As always, the details get messy, but there are two ways that small RNAs can work. - One is the siRNA (short interfering) pathway, which leads to mRNA destruction. - The second is the miRNA pathway that leads to translational repression.
Secondary structure effects
- Secondary structure near the 5'-end of an mRNA can have either positive or negative effects - Hairpin just past an AUG can force a pause by ribosomal subunit and stimulate translation - Very stable stem loop between cap and initiation site can block scanning and inhibit translation
Review lecture 15
- We've followed the birth and processing of the protein coding mRNA - We've learned about the biogenesis of the protein synthesizing ribosome - We've covered the creation and processing of tRNAs, snRNAs, snoRNA etc. - One last step before we can cover translation.
mRNAs are produced by RNA polymerase II:
- mRNAs are capped, spliced, modified and polyadenylated - Eukaryotic mRNAs are exported from the nucleus - NMD and other quality control processes regulate these transcripts
miRNA biogenesis
- miRNAs are encoded in genes in myriad ways pol II transcript is capped; long spliced rna stitch several rnas thats capped (multi-micron less introns) miRNA protein coding gene; exons put together 3' UTR protein coding genes can reach a functional miRNA miR-35 to miR-41
Germ-line defenses
- piRNAs are there to prevent these elements from moving at a time critical to development. Why is this important? --> modifying genome at this time is fixed in the germ line; then bad things can happen - Any genomic modifications at this time will be fixed in the germ-line... Bad things happen when these elements move around. ESS
Mature tRNA structure
- tRNAs broadly are the most abundant RNA transcripts in cell (~25M/cell in humans) - tRNAs are highly modified (avg 13 mods / tRNA) - tRNAs fold into highly similar shapes - Yeast encode 275 tRNA genes - Humans encode 497 tRNA genes + >300 pseudogenes - if the box is blank they are not conserved -if the box has a letter in it then it's highly conserved
transposon movement
1) transcription Transposon copied from DNA, creating a mobile transposon 2) RNA processing This MT is then inserted into DNA in new location 3) RNA export 4) Translation 5) Post-translational modifications and RNP formation 6) Entry into the nucleus 7) TPRT 1, 2, & 7 happen in the nucleus 3, 4, 5, & 6 happen in the cytoplasm
You have a patient that presents with what you suspect is an autoimmune disorder (AI), but tests negative for lupus and Sjögren syndrome. - Excited to use your molecular biology knowledge, you approach this as a novel AI disorder. 1. What is the nature of an AI disorder? 2. How can you go about determining what the autoantibody is reacting with? 3. How will you determine the interacting components of the autoantigen? For this exercise, you should assume that you can purify and characterize antibodies, antigens and cell types
1. & 2. perform a western blotting: You would take the cells that are providing antigen; break them open; do a total lysed when you transfer this to the paper we do the western blotting on we can determine the molecular weight of that protein by using serum from the patient. - you figure out the polypeptide is a 50 kD protein. 3. Use blood from patient to make a serum and use chromatography: Get the antibody from the serum & cross-link it to the beads in the chromatography chamber then all the proteins used for the total lyse is put through it, wash away the extra proteins which are not bound to the antibodies & place on new gel to then elude out the ones specifically bound to the antibodies and those would be the TARGET protein. can run an RNA gel (rRNA binding proteins) mass spectrometry & protein sequencing & the western blot can tell us that we have found the target protein that has been identified.
EF-Tu cycle (accommodating the A site) - first GTPase cycle
1. Binding of aminoacyl tRNA: aminoacyl tRNA binds to A site, escorted by EF-Tu bound to GTP. During tRNA binding, the GTP is hydrolyzed & EF-Tu is released. EF-Ts helps recycle EF-Tu. GTP bound form binds to amino acid and lands on GTPase stalk & is testing hydrolyze GTP to GDP leads to structural rearrangement that leaves tRNA in that site and ejects something out of the ribosome. if EF-Tu amino acyl tRNAs dont fit, it'll try again until it find one that will pair. Ribosome = GAP GEF 2. Peptide bond formation between carboxyl group of fMet (or, in later cycles, of the terminal amino acid) at the P site & amino group of the new amino acid at the A site. 3. Translocation: mRNA advances by 3 nucleotides, peptidyl tRNA moves from A site to P site & the empty tRNA moves from P site to the E site; hydrolysis of GTP to GDP happens WASH, RINSE, REPEAT
tRNA processing in E. coli
1. Endonuclease cuts off the 3' end loop 2. RNaseD (exonuclease) removes these 7 nucleotides one by one (blank) 3. RNase P (between U & G) 4. RNase D removes these 2 nucleotides (U & C) 5. 4-Thiouridine UU O^2-Methylguanosine G 2-Isopentenyladenosine A Pseudouridine U Pseudouridine U
How does this happen? (tRNA)
1. Nuclease - 2 cuts made on tRNA, OH5' and 2'-3' P 2. Folding - OH 5' left 3. Ligase - perfectly good functional tRNA
A lesson in perseverance
1. Petunia pigments normal pigment --> incr. the flower pigment gene expresion will result in less pigment (molecules) of that color 2. Wiggling worms: induce the suppression of a phenotype par-1 mRNA 5'-------3' antisense RNA 5'------3' 3'-----5' eliminated RNA from the cells Others were using antisense RNAs to shut down the function of cellular RNAs in worms. Systemic Spreading 2 ways to do experiment: soaking or feeding OR microinjection into the intestine 3. Developmental timing of general gene expression What do they have in common?
If there are only a maximum of 64 possible combinations, why encode so many?
1. TIME: more gene expression in embryonic stem cells and embryo cells during development than we do as adults 2. SPACE: brain, skin cells; all cell types have this constellation of different genes that are going to be expressed. EX. So, if you have in the brain, expression of a tRNA for leucine that is different tRNA for leucine that's expressed in your pancreas... Now you have 2 different genes for the same tRNA BUT you need both of them. ONLY one of them will be transcribed in the brain & the other will be transcribed in the pancreas. So now you need 2 genes for that leucine tRNA. 3. DOSAGE: 20 amino acids encoded by 500 tRNAs. EX. looking across human proteome - add up all the times you translate a leucine (25) codon; lysine (30) & phenylalanine (2) - leucine and lysine are abundant in human proteome. - you don't need an equal amount of tRNA that are being made for all 20 aa and in that case we can utilize increased gene dosage for that. Each have their own promoter & they can be recognized *** We can see where we need those extra tRNA to make sure there's enough to decode all those specific aa codons.
Ribosome resistance
1. What might the basis for antibiotic resistance be? 2. What is being done about it? 3. Why don't we see new antibiotics on the market very often? 4. What can be done about it?
Aminoacylation of tRNA steps:
1. amino acid + ATP ---> aminoacyl-AMP + PPi 2. aminoacyl-AMP + tRNA ---> aminoacyl-tRNA + AMP
2 reactions of aminoacylation of tRNA
1. bind to amino acid with specific R group 2. activate amino acid with AMP 3. Bind to the tRNA 4. transfer amino acid onto active site
What are 2 different RNA polymerase III promoter recognition sites?
1. tRNA gene 2. 5S RNA gene
Structural basis for inosine usage (wobble base pair)
34 = I I * C I * A one nucleotide can base pair with A and C anti-anti syn-anti
tRNA mechanism
5' exon1--intron--exon2 3' 1. Two endoribonucleases (endonuclease) cut the pre-tRNA 2. Exon 2 has its 5' OH that turns into a phosphate repaired *energy is required from a GTP* 3. Exon 1 gets a 2'-3' cyclic phosphate cyclic 2'-3' phosphodiester bond --> opened up to make a 2' phosphate with an OH kinase- takes GTP; transfers gamma phosphate onto 5' OH to form a 5' phosphate after reaction is done. *Ligase has 2 steps: adenylation & linkage* 4. Exon 2 is adenylated: A placed on like a cap, p-p 5'-3' phosphodiester bond is formed 5. Exons are ligated together 2-phosphotransferase--> 6. 2' phosphate is removed, creating a perfect tRNA
RNA polymerase III promoter recognition sequence for tRNA:
A box & B box
Transposon defense in some budding yeast
A budding yeast fungi pathogens for humans, corns B AGO1 DCR1
Phenotype without genotype!
A non-silenced plant (black leaves) grafted onto a silenced plant (white leaves) [eventually will attain phenotype without changing genotype] induces rapid silencing within the graft.
Different means to the same ends: IRES elements come in a wide variety of shapes and sizes!
A. poliovirus C. hepatitis c B. Encephelomyocarditis virus (EMCV) D. Cricket paralysis virus
siRISC (siRNA induced silencing complex)
AGO2 = argo knot 2 can figure what if perfectly base paired or complementary can lead to 5' OH end leads to cleavage at 5' and 3' end (destruction machinery)
Adaxawhat?
Abaxial: underside of the leaf (concave) Adaxial: top side of the leaf (convex)
tRNA synthetases: Class I
Acceptor stem-pink: many of the peptides are here anticodon loop-pink: same RNA sequences will be here. we can get one enzyme interacting with multiple different substrates. editing site -pink activation site ex. LEU all those diff ones will have identity elements
Initiating translation in prokaryotes
Addressing the message: Shine-Delgarno sequence
You are designing new antibiotics that you wish to inhibit translation only in prokaryotes. You notice that these new compounds are spectacular drugs, with no resistant mutants arising by mutation of the rRNA or proteins, and no enzymes in nature seem to be able to metabolize these antibiotics. You experimentally validate that these drugs occupy the P-site in the ribosome and that they H-bond with universally-conserved nucleotides. Explain why no resistance mutations arise (5 points) and why these are NOT likely to be safe for humans to take (5 points).
An effective inhibitor must be specific to the (set of) organisms that one wishes to kill. The most potent inhibitors of translation contact the most important nucleotides and proteins of the ribosome for structure and chemistry. If the drugs bind to universally conserved nucleotides, these are most likely the ones required for function, so mutants that prevent binding of the new drugs will alter structure and function such that they will not both escape the drug and still perform peptide bond formation. They are not likely to be useful for human use, since humans have the same arrangement in the active site of the ribosome - and universally conserved nucleotides for structure and function will be required in humans as well.
initiation of translation
Another opportunity for regulating gene expression; most commonly at hte initiation stage; initiation of translation of some mRNA can be blocked by regulatory proteins that bind to specific sequences or structures within the ultranslated region at the 5' cap and the poly-A tail of an mRNA molecules FINISH REVIEWING
RNA polymerase III promoter recognition sequence for 5S RNA:
C box
Cytoplasmic processing of miRNAs
Dicer creates miRNA duplex, dicer selects one strand to make miRNP that helps in translation
How dose EF-Tu work?
EF-Tu/GTP/tRNA constantly samples the A site codon Affinity of EF-Tu for GTPase stalk on 50S Affinity of tRNA for mRNA codon Hydrolysis of GTP initiates a dramatic conformational change in the protein
Anatomy of the translating ribosome
EPA APE A = aminoacyl site ¤(aminoacylated tRNA freshly loaded into ribosome) P = Peptidyl site ¤Where the action happens E = exit site ¤tRNA that is about to exit the ribosome mRNA is sandwiched between the SSU and the tRNAs mRNA is sandwiched between the SSU and the tRNAs
Differences in initiation mechanisms
Eukaryotic ¤Begins with methionine ¤Initiating tRNA not same as tRNA for interior ¤No Shine-Dalgarno ¤mRNA have caps at 5'end Bacterial ¤N-formyl-methionine ¤Shine-Dalgarno sequence to show ribosomes where to start
Eukaryotic initiation mechanisms
Eukaryotic 40S ribosomal subunits locate start codon by binding to 5'-cap and scanning downstream to find the 1st AUG in a favorable context
Whats happening in autoimmune diseases?
HLA: human leukocyte HLA-ClassII: Exogenous antigen: creates proteins cells will create small peptides (green diamond) then its exposed to the extracellular surface (alpha 1,2 Beta 1, 2) works to expose that peptide to the immune system. Amplification of the B cells and T cells can ward off that infection, virus, etc. HLA-ClassI: Endogenous antigen: Same process of chopping up protein and using the small peptides to expose on the surface. The peptide is saying that its okay and that its "us". A disease happens when HLA-ClassI and HLA-ClassII get MIXED UP. Cells self-identify through HLA (MHC) Class I - if self-peptides are presented in HLA Class II, they are recognized as foreign and stimulate an immune and inflammatory response.
Aminoacylation of tRNA
How do tRNAs help direct the synthesis of proteins? - Recall the CCA-3' - This nucleotide has an amino acid attached to it. A class of enzymes call aminoacyl tRNA synthetases (aaRSs) catalyze this reaction in a tRNA-specific manner. - This reaction happens in two steps. First, the amino acid is adenylated by attaching to an ATP (via an AMP) molecule. Next, this activated amino acid is covalently attached to the proper tRNA. One aaRS per amino acid - even if multiple anticodons! What does this mean for selectivity? Both steps happen in the same enzyme.
Mechanism of IRES function
IRES adopts a P-site tRNA conformation!
Autoimmunity: creating an inappropriate immune response against "self"
Immunity against self is generally prevented by selecting against antibodies that are self-reactive Autoimmunity arises when that selection goes wrong. if you aren't able to eliminate B cells and T cells Without getting too far into the immunology weeds...
Detailed view of initiation in eukaryotes
Initiation begins at the cap, in the context of the mRNA and 40S subunit Scanning goes from the cap to the START codon that conforms best to the Kozak rules GTP is hydrolyzed by eIF5, triggering eIF2 to bind to GDP and releases a set of initiation factors 60S now binds and creates the 80S ribosome for protein translation. **Components very much not to scale....
Transposable elements in the human genome
LINEs 21% SINEs 13% retrovirus-like elements 8% DNA transposon fossils 3% Only about 10 active L1 elements in the genome...
Lupus and other autoimmune diseases
Lupus erythematosus, Sjögren syndrome and other autoimmune diseases result from inflammation due to inappropriate production of antibodies to RNP components such as the U snRNPs.
Some miRNAs are encoded in several copies
MIR156 same targeting a-f are encoded in different genes why not encode all of them in one gene transcript? Is this a dosage issue?
tRNA (transfer RNA)
Modifications can confer: Structural stability Enhanced base stacking Protein recruitment Induce interaction by + charge Wobble flexibility Enhanced translation function
tRNAs have introns
NO Group I, Group II or spliceosomal introns *requires different enzymes & completely different mechanisms* Their removal requires an endonuclease, a kinase, a ligase and a 2' phosphotransferase ¨Why?
Consider the differences between similar amino acids: valine, leucine, & isoleucine
One methyl group is all that is different in some cases. tRNA structure is also very similar between different molecules - E. coli tRNAphe - Yeast tRNAmet - Human tRNAlys3
MicroRNAs
Other small RNAs (21-23 nt) called microRNAs or miRNAs lead to translational repression, but not mRNA destruction. How do these RNAs operate? --> repression or destruction How are they created and processed? miRNA biogenesis How is this whole system regulated?
Editing of mischarged tRNAs
Protein binds to aa, has a specificity for both tRNA & aa. The aa is bound in the AS (acetylation site) and binds to ATP then it transfers the acid; it measures the aa twice before hydrolyzing AMP to start the process all over again. - Mischarging can happen in up to 1% of the cases naturally. - With editing, that rate is decreased 2 logs to 1 in 10,000 cases. - This seems to be the limit of tolerance for translation fidelity too... - How does editing work? Back to the puzzle pieces!
tRNA Biogenesis: RAN (protein), GTPase, How does it work?
RAN: shuttling protein things are shuttled nucleus to cytoplasm on right and vice versa on the left Ran-GTP state: has affinity for exportin-xyz (adapter) binds to CRM1 [protein - NES] Expt binds to this protein Exp-t will bind to tRNA that'll bind to CRM1 and that complex will bind to Ran-GTP --> engage with nuclear pore and be excised out of nucleus. (into the cytoplasm) GAP (on cytoplasm side): undergoes structural rearrangement that will no longer bind to the cargo so they will dissociate from one another - GAP hydrolyzes GTP to GDP Ran-GDP: affinity to importin (adapter) alpha and beta --> engages with nuclear pore & goes into nucleus - imports proteins into the nucleus GEF (on nuclear side): RAN replaces GDP for a GTP using RCC1 to continue cycle & balance everything out NES: nuclear export signal - exposed in the nucleus & tucked away in the cytoplasm NLS: nuclear localization sequences -tucked in on the nucleus side and exposed in the cytoplasm
Recognition of a STOP codon
RF1 & RF2 is recognizing different groups of nucleotides how to recognize stop codon with a peptide? interactions with U1, A2, A3
Chromatin modification in fission yeast
RITS: RNA Induced Transcriptional Silencing chromatin in centromeres and telomeres Production of small interfering RNAs builds in localized RNA system - in regions we want to enforce heterochromatin
7SL SRP RNA
RNA Pol III transcript
Structure of pri- and pre-miRNA
RNA gene is processes into pri-miRNA i cut out by nuclease called Drosha, Pasha (nuclease) and is cleaved as 5' 3' end then its a pre-miRNA and then goes to cytosol from nucleus. pre-miRNA are cleaved on both ends by Dicer (nuclease) and made into miRNA/miRNA* duplex then it becomes a miRNA and results in a miRISC
Spinal Muscular Atrophy - an RNP disease
SMA is an autosomal recessive condition resulting from a defective SMN gene resulting in improper Sm snRNP biogenesis interupts snRNP biogenesis
Phylogenetic conservation of SRP (protein)
SRP = signal recognition particle recognizes the signal sequence. ffh nad SRP54 is recognized to feed into regulatory pathway
Miscellaneous stable RNAs
Several RNA Pol III transcripts have unique biogenesis pathways U6 snRNA is an RNA Pol III transcript with a unique cap structure
In all cells, but eukaryotic cells in particular, proteins need to know where to go.
Some proteins need to go to the nucleus, remain in the cytoplasm, to the mitochondria, and others need to be targeted to the plasma membrane or for secretion. starts during translation How does this happen?
How do snoRNAs become snoRNAs?
Some snoRNAs are independent transcripts But many snoRNAs are encoded within mRNA introns
SMN
Statement of Medical Necessity
What does TFIIIC do?
TFIIIC binds to the A & B box sequences & helps to position RNA polymerase properly after the binding of TFIIIC, we have the binding of TFIIIB; which is a tri mer of proteins to help RNA pol III identify the start site of transcription at nucleotide #1
Antiviral chemotherapeutics (ex. experiement against HIV)
Take stem cells out from a patient, then transduce pluripotent immune cells; put them back into the patient and then try to create cells that had an anti HIV miRNA in them. Turned out the cells did this and became resistant to HIV infection but the problem was that the virus mutates very rapidly. so it didn't work that well.
7SK RNA
The only known RNA to be involved in eukaryotic RNA transcription Binds to PTEF-b (a general TEF) and inhibits its function
tRNA modifications
There are some modifications of ALL tRNAs The modification landscape across phylogeny demonstrates the flexibility and evolution of these processes The modification landscape across phylogeny demonstrates the flexibility and evolution of these processes
How do these antisense RNAs work?
Turns out the operative RNAs are very very small. (microRNAs) This allows them to evade the surveillance in the immune system. Short RNAs that lead to mRNA destruction are called siRNAs or Short Interfering RNAs
proteins that code for translation to stop
UAA UAG UGA
Phylogenetic distribution of RNAi
Unlike spliceosomal introns and snoRNAs, the RNAi system is not present in all eukaryotes. Additionally, the RNAi system seems to have vastly varying functions in different organisms. DNA repair and other homeostasis interactions How? Why? Stay tuned!
Chromatin remodeling function of RNAi is conserved
Will we need to memorize any of these?
take on down, pass it around
a -hybrid states formation-> b -EF-G binding-> c -GTP hydrolysis conformational change; translocation ratcheting-> d -EF-G dissociation -> e f <--> f
The RNases used in the process
a Pri-miRNA processing by Drosha terminal loop determines where the cuts are made there are 2 cleavage events anchors at the terminal loop & cleave downstream from that b Pre-miRNA processing by Dicer anchor at cleavage event then you measure up towards the terminal loop to cleave and get the double stranded microRNA (dsRNA stem ~ 22 nt)
Eukaryotic RNase P (RNP)
a little more structural - evolutionarily conserved structures contains several proteins RNase P = ribozyme !! RNase D = exonuclease that degrades the second step
codon
a sequence of 3 bases in mRNA that code for a particular amino acid or for chain termination
Antibiotics
a) antibiotics targeting the 30s subunit; Antibiotics targeting the SSU bind near the important parts b) antibiotics targeting 50s subunit. c) antibiotics at the PTC. Antibiotics targeting the LSU bind near the important parts Fusidic acid inhibits EF-G Tetracycline (blue) Pactamycin (green) Hygromycin B (red) Hygromycin DEFORMS the decoding center: H44 & H45 Pactamycin binding deforms the mRNA path Antibiotics that target the PTC (peptidyl transferase center)
miRNAs have tissue-specific expression
a) homo sapiens lung kidney brain - high let-7 RNA expression
asRNA
antisense small RNA-allows these molecules to bind target mRNA which can either stabilize the target mRNA or make it susceptible to degradation
some amino acids
are specified by 2 or more codons
Genetic code
based on the sequence of bases along a nucleic acid
elongation (translation)
begins by the appropriate aminoacyl-tRNA binding to the codon in the A site of the ribosome.
tRNA biogenesis
beta-globin: -100 Glucocorticoid receptor: -1000 Histone H2B: oct-150 start side of transcription is upstream from A and B box
Human therapies based on RNAi
clinical therapies - used in specific diseases because its cleared out by the liver relatively quickly mostly ocular diseases
RF3 looks like other ribosomal GTPases
confirmation change helps to cleave polypeptide
Animal miRNA functions
element instabilities markers for cancer by looking at the concentrations or the prognosis bases on the presence of particular mRNAs
endonuclease vs exonuclease (prokaryotes & eukaryotes)
endo - cuts inside nucleic acid (you dont need a 3' or 5' end hydroxyl exo - cuts ends outside the RNA (will latch onto 3' or 5' OH to munch on one nucleotide at a time.
1. How does one active site accommodate two different nucleotide substrates? 2. How does the enzyme only add in the sequence 5'-C-C-A-3'?
exonuclease cleaves off terminal Us, CCA adding enzyme doesn't need a template - uses affinity for nucleotides (cytosine and adenine 1. ARRANGEMENT OF AMINO ACIDS IN THE ACTIVE SITES A73 is the last nucleotide strand before process happens C74 is the first nucleotide addition RNA is changing shapes - accommodating new tRNA with 2 nucleotides.
RF3 and RRF
help dissociate the ribosome
Closer look at elongation (right after translation initiation)
how GTPase work in elongation: Initiation: fMet (peptide) E site = empty P site = middle tRNA site where the aa is inserted A site = empty
synonyms (multiple codons for the same amino acid)
in most cases differ only in the 3rd base Similar codons tend to code for similar amino acids. *effects of mutation are minimized
Plants use RNAi for antiviral defense
infect plant with ring spot virus creates a phenotype in some plants they got the elimination leaves above a certain position were resistant to virus in plant sequence-specific nuclease initiate RISC viral RNA immunization shot plants used RNAi for antiviral defense
Translation from beginning to end
initiation elongation termination
stabilization of tRNAs by CCA ends
keeps a thermodynamic grip; terminal transferase reaction placed on the CCA end
A monkey wrench in mammals
long dsRNA signals organism something is wrong (virus) --> initiates pathway which activates interferons. interferon production will activate the immune system and kill cells that contain long dsRNA. - protein synthesis stops - all mRNA is degraded - cells die
Plants have a much more complex RNAi system
lots of ways to attack pre-mRNAs all nucleases have the same function (Dicer)
SRP cycle
mRNA is translated; new polypeptide w initial signal peptide; sRP bound to signal peptide; SRP bins to SRP receptor (GTPase); once its docked properly, the growing polypeptide chain will be fed & the protein signal peptidase is cleaved off; SRP is liberated. signal sequence is removed from growing peptide. growing peptide turns into complete peptide.
We have studied the means of production of the ingredients for protein translation:
mRNA, ribosome, & tRNA
miRNAs are evolutionarily conserved (not all)
miR1: present in 2 round worms, fruit flies, & humans the sequence is almost identical & preserved thru evolution due to importance
miRISC (miRNA-induced silencing complex)
miRISC can lead to either mRNA repression or to destruction indirectly by targeting them to the P-bodies in the cytoplasm P-body mediated destruction si or miRNA can regulate their targets
A RISC-y proposition
miRNA duplex pathway: targeted and has 2 different outcomes 1. translational repression siRNA duplex pathway: 2. mRNA cleavage you can have one type of miRNA that can perform both
Battle of the Bulge (functions)
miRNA: translation repression (repress) siRNA: mRNA cleavage (cleave)
Initiating translation in eukaryotes
no shine-Delgarno sequence, but it scans for an AUG codon near the cap the initation factor (eIF5) in 80s ribosome
Non-templated NTP addition
nucleotidyl transferase reaction: adds the CCA - putting 3 nucleotides on instead of 1
termination of translation
occurs when a stop codon in the mRNA reaches the A site of the ribosome
Processing and export of miRNA
pri-miRNA that will be cleaved by Drosha to create pre-miRNA and is exported by exportin 5
The elongation cycle
regulated by 2 GTpases: EF-Tu-GTP and EF-G-GTP
Plants use RNAi for development
regulation miRNA are over expressed and consequences were observed what the miRNA is normally doing target family: transcription factors Abaxial leaf fate you can control the size and the shape of leaves and flowers of plants (speciation, hybridization) - all controlled by miRNAs 6 vegetables that are the same plant: broccoli, cauliflower, khale, cabbage
tRNA discrimination
relies on those elements that differentiate the tRNAs to a degree sufficient to allow discrimination Editing helps in those situations that are close enough at the amino acid or tRNA level to allow confusion
What does it say about the need for RNAse P proteins if protein does not make up the active site?
requires the RNP (ribozyme) to cleave much more, more quickly
Chemistry of peptide transfer
ribosome = ribozyme
RNase P (RNP)
ribozyme that processes tRNA precursors & cleaves the 5' end of pre- tRNAs. active site is made of nucleic acids
Ribotoxins
russian spy followed british spy umbrella. hit with ricin poison and started destroying ribosomes. ¨It was a dark and gloomy night... castor beans - ricinus communis ---> ricin. Chains pulled into cells; they have an endonuclease function; nucelotide important for elongation. cleaves 1 nt in the large subunit RNA. SRL = sarcin ricin loop (1 nt change bc of 1 evolutionarily conserved nt) will cause ribosomes to kill themselves
Signal sequences
signal peptide is made up of Hydrophobic amino acids (8 or more) at the very amino terminus of a nascent polypeptide recruit SRP
siRNAs
small/short interfering RNAs, turn off gene expression by directing degradation of selective mRNAs and the establishment of compact chromatin structures
Constipating the ribosome
structures bind to the exit tunnel. ex. Erthyomycin (ERY),
Many other RNAs exist in the cell
tRNA (translation) snRNAs (pre-mRNA splicing, transcription) snoRNAs (ribosome biogenesis) scRNAs (functions unclear) miRNAs (regulate gene expression post- transcriptionally) Telomerase RNA (telomere maintenance) SRP RNA (protein secretion) 7SK RNA (transcription regulation)
Archaea have introns too
tRNA endonuclease in archaea tRNA endonuclease in eukarya
GTPase landing strip
the function of GTPase stalk (pink) is to feed into the entry tunnel of the ribosome, can shove tRNA over or help to determine if translation/ termination needs to happen GTPase lands on a stalk complex: bacterial/ /eukaryotic
Some miRNAs are temporally regulated
they are only present during specific time points during development a) Drosophila melanogaster b) Danio rerio
Some plant viruses have developed anti-RNAi strategies
they have proteins inside of them; at the bottom are the RNA duplexes that are acted on by RISC, at the top they are like sponges. hosts will develop strategy to prevent RNAi from hurting them. plant warfare.
tRNA processing in S. cerevisiae
transcript from intron-containing gene: splicing occurs transcript from intron-less gene: no splicing Both go through: 5' endonucleolytic removal of 5' leader - RNase P 3' processing - Rex1, RNaseZ, Lph1 3' CCA addition - Cca1 exports out to the cytoplasm
CCA end of tRNA
universal in all tRNA in nature which is important for putting an aa on that end
miRNAs often operate at multiple sites
when they bind to an mRNA they are regulating a) incorporated into RISC b) there are 7 different binding sites; done by base pairing redundancy has several different functions - typically in 3' UTR; also typical site of mRNA binding
piRNAs - PIWI interacting small RNAs
¤P-element Induced WImpy Testes ¤Discovered in Drosophila, conserved in mammals ¤Expressed only during spermatogenesis and oogenesis ¤Biogenesis pathway is completely different ¤Function is to repress transposon / repetitive element movement during gametogenesis and embryogenesis
Prokaryotic translation termination is mediated by 3 factors:
¤RF1 recognizes UAA and UAG (2/3) ¤RF2 recognizes UAA and UGA (2/3) ¤RF3 (like EF-Tu) is a GTP-binding protein facilitating binding of RF1 and RF2 to the ribosome
Eukaryotes have 2 release factors:
¤eRF1 recognizes all 3 termination codons ¤eRF3 is a ribosome-dependent GTPase helping eRF1 release the finished polypeptide
critical questions
¨How to ensure limited "off-target" effects? 100 or 1000s of RNA; how to make sure the one you are designing is only targeting the chosen one. ¨Which regions to target? 3' UTR; imperically determine whats best. ¨Viral and cancer evasion by mutation ¨Be careful what you ask for!
Anatomy of the polypeptide exit channel
¨Think about that channel - it must accommodate all types of amino acids - how does it do that?
2 classes of tRNA synthetases
•Class I aaRSs: Identity elements usually include residues of the anticodon loop & acceptor stem. •Class I aaRSs aminoacylate the 2'-OH of "A" at tRNA 3' end. •Class II aaRSs: Identity elements for some Class II enzymes do not include the anticodon domain. •Class II aaRSs aminoacylate the 3'-OH of "A" at tRNA 3' end.
