Cell Biology Exam 4 Study Guide

Why is the Carboxy Terminal Domain (CTD) of RPB1 (Pol II) so important? (Fig. 9-15, Fig. 10-12, and lecture slides).

- Seven amino acids stretch repeated about 26x in yeast, 52x in mammals - (Tyr - Ser - Pro - Thr - Ser - Pro - Ser)26-52 - Tyr and Ser residues will be phosphorylated during transcription elongation. - the un-phosphorylated form is needed to assemble transcription initiation complex before transcription starts. - Phosphorylating the tail of RPB1 may be the trigger to begin transcription once the complex is assembled

What processing steps occur to a tRNA? (Fig. 10-45). What's RNAse P composed of, and what does it do? T/F. Splicing of pre-tRNAs uses the spliceosome (snurps 1, 2, 4-5-6).

- pre-tRNA with a 5' leader sequence that needs to be cleaved off -CCA- placed on every tRNA (The OH group forms the ester linkage with the amino acid when it becomes charged) Factoids: - RNA Pol III - some have introns where the anti-codon will form. First event in tRNA Processing: - 5' leader sequence is removed by RNase P: (a ribonucleoprotein endonuclease Made of an RNA molecule and a protein) - in E. coli, RNase P consists of a small protein and an RNA molecule, called M1 (ribozyme with enzymatic activity in vitro, under high [Mg+2]; can cleave the 5' leader without the its associated protein component. - M1the protein simply increases the rate of cleavage in vivo at physiological [Mg+2]. In vivo = living cell RNA acting as the enzyme Nucleotide modifications: - 10% of the pre-tRNA nitrogenous bases are modified 1) 3' uridines replaced by CCA. 2) methylation of some purine bases 3) isopentenyl group added to some purine bases 4) conversion of some uridines to - pseudo-uridine (ψ) - pre-rRNA - dihydro-uridine (D) - ribothymidine (T) Pre-tRNA "Splicing" 1. One step intron removal using an endonuclease. - Endonuclease recognizes borders of intron and cleaves in one swoop 2. Leaves a 2', 3' cyclic mono-phosphate (unique to endonuclease that made the cleavage, red arrow). 3. Process catalyzed by proteins (not RNAs). RNA Ligases. a) links 2 exons to give the mature tRNA 4. Process is conserved. can have slight differences in species

The steady state concentration of a mRNA depends on its rate of ______________________ and its rate of _____________________________.

- synthesis - degradation. - Referred to as half-life. - Different mRNAs have different half-lives (dependent on the rates of synth. and decay).

cis-acting DNA enhancers and promoter proximal elements. (Fig. 9-23).

1) Biochemical identification and characterizations: - cis-DNA elements (enhancers), once obtained, were used to identify/isolate the "cognate" trans-acting factors (proteins). · Protein factor specifically binds to the enhancer element (major groove) · Cis elements attach to the column for affinity chromatography · What about the protein factors that bind these enhancers and promoter proximal regions? How did they identify and characterize them? The protein factors were once called 'trans' factors that could bind to the "cis" DNA elements. · Steve McKnight who invented the Linker Scanning used the enhance DNA sequence itself to affinity purify the protein that would bind to it. He called his protein CEBP (Cellular Enhancer Binding Protein). He isolated the protein initially form rat liver, sacrificing 100's of rat to get enough of the protein.

Stop-transfer sequence, signal-anchor sequence, and multi-transmembrane insertion (Figs. 13-11 and 13-12a, replacement for Fig. 13-14).

1. Stop Transfer Anchor (STA): a. Serves as a membrane anchor i. Becomes the trans-membrane alpha helix (red) ii. Ex. Insulin receptor 1. The alpha helix has to span the membrane and move laterally out of the translocon 2. Appears to be a normal secretory protein a. Has a signal sequence at very amino terminal end and it interacts with the SRP and the SRP interacts with the LSU ribosome to anchor the ribosome to the translocon iii. When the stop transfer anchor is translated it serves as the transmembrane alpha helix 1. Any insertion of this protein through the translocon into the lumen of the rER stops. 2. Alpha helix spans the membrane a. moves laterally out of translocon i. translation continues now producing the carboxyl end of the protein ii. carboxyl end remains in cytosol and the amino terminal end lacking the signal sequence remains in the exoplasmic space (lumen of rER). 1. Proteins with opposite orientations: i. N-terminus on the cytoplasmic side ii. C-terminus on the exoplasmic (luminal) side b. These proteins have a single internal Signal Anchor (SA) topogenic sequence. (That is not cleaved) c. Dual function (SA serves as both signal and anchor) 1. Signal sequence - not at very beginning of protein so the N-terminus has already been translated by free ribosomes before it even emerges from the ribosome a. Once the signal sequence emerges, the Signal recognition particle recognizes the signal sequence and the large ribosomal subunit and delivers it to the SRP receptor much like before for secretory proteins. b. The ribosome is then delivered to the translocon and notice the amino terminus is already in the cytoplasm 2. Signal Anchor sequence now serves its anchor function, the trans-membrane alpha helix, anchoring the protein in the membrane, further translation will produce the carboxyl end of the protein which is pushed into the exoplasmic luminal space of the rough ER. a. The protein now has the reverse orientation. 2. Multi pass proteins: a. How are multi-pass proteins inserted? i. Alpha helix to span each pass b. These proteins are common for ion channels c. Each alpha helix is thought to be a topogenic sequence i. For ex. First alpha helix acts as a signal-anchor (SA) 1. Dual function - first as a signal then as an anchor 2. SRP interacts with the first alpha helix 3. SRP receptor and translocon are also involved ii. The second alpha helix however serves as a stop-transfer anchor (STA) iii. Translocon used to position the 2 alpha helices in the membrane d. Subsequent pairs of alpha helices will require the translocon but we will no longer need the SRP or SRP receptor e. The second pairs of alpha helices, alternating between signal anchor in red and stop-transfer anchor in blue, will insert into the membrane with the help of the translocon, i. Goes until all alpha helices are placed in the membrane. f. Amino terminus in cytoplasm and carboxyl end in cytoplasm i. What about G-protein coupled receptor: 1. Amino terminal end in the exoplasmic luminal space and the Carboxyl end in cytoplasm 2. First, an even number means both ends of the protein end up on the same side 3. Like GPCR - odd number of alpha helices will make the ends end up on different sides of the membrane. 4. The GPCR the amino terminus was on the exoplasmic luminal side a. Meaning the GPCR had to start with a signal-stop transfer anchor signal first i. Opposite orientation of the topogenic sequences in a given pair. How are multi-pass proteins inserted? · First and all subsequent odd number helices act as SA's; o all even numbered helices act as STA's. In other words - the third alpha helix acts as a SA - the fourth alpha helix acts as another STA · But helices 3 and 4 insert into the membrane as a hairpin (pair). - SRP and the SRP receptor do NOT play a role here - but the translocon is still involved (somehow). Instead of using an alpha helix, - some proteins are anchored to membranes by § Glycophosphatidyl inositol (GPI) anchor - Protein inserts as previously described. - Endoprotease (transamidase) cleaves the protein in the exoplasmic space and transfers it to the GPI anchor § the lumenal portion to GPI. o Protein remains in the exoplasmic luminal space but this will become the outside of the cell. -Perhaps becomes a portion of the plasma membrane the exoplasmic luminal space of the ER emerges as the outside of the cell.

Who the heck is Günter Blobel? Simple question → elegant answers → Nobel Prize.

1999 Nobel Prize. How does the cell sort them to their correct locations? o membrane proteins o mitochondrial proteins o chloroplast proteins o nuclear proteins o lysosomal proteins o extra-cellular matrix proteins Process of protein targeting or sorting: - signals (zip codes) and sorting events. - signals = short amino acid motifs

3' cleavage and polyadenylation (Fig. 10-15): What's AAUAAA? What's the G/U rich region? Where's the cleavage site? What's polyA polymerase doing?

3' Cleavage and Polyadenylation: · All mRNAs except the histone mRNAs have Poly(A) tail o Also, histone pre-mRNAs do not contain introns · Poly(A) signal sequence within the 3' untranslated region (UTR) o -AAUAAA- signal sequence o Signal sequence lies 10-35 nts upstream from where the poly(A) sequence will be added o Before cleavage, notice further downstream is another Poly(A) signal sequence § Assists enzyme complexes to form and cleave for Poly-adenylation o G/U rich region further down stream o This G/U sequence is cleaved off so poly(A) tail can be added o The G/U rich is another Poly (A) signal sequence Factors in Cleavage and polyadenylation: · CPSF: (cleavage and polyadenylation specificity factor) o 4 different proteins o They bind to the first poly(A) signal (AAUAAA) o Complex is about 360kDa · CStF: o Binds to second poly(A) signal sequence § The G/U rich sequence · CFI and CFII o Cleave the mRNA in the 3' untranslated region o Poly(A) polymerase tacks on 200-250 Adenine nts to the freshly cut site within the 3' UTR o The assembly of all the proteins is cooperative - one binds the other bind as well o Once you have the Poly(A) tail, you have proteins that bind to the tail. § No naked RNA in the cell o Inside the nuclease poly A binding protein II will coat the poly A tail o Once mRNA in cytoplasm then poly A binding I will decorate the Poly(A) tail. Discrete sites within the nuclei of eukaryotic cells for polyadenylation and splicing: · The eukaryotic nucleus has sites where polyadenylation and splicing are rapidly occurring.

The different classes of DNA-binding domains (lecture notes and pages 308-311 in the 7th edition, but pp. 384-386 in the new 8th edition) (Fig. 9-30a-d). - combinatorial control (Figs. 9-32, 9-33a. 9-34) - enhanceosome (lecture figure)

A closer look at the DNA-binding motifs/domains often found in eukaryotic transcription factors · Most contain alpha helices, commonly used to bind the major groove if B form DNA · Atoms within the proteins make hydrogen bonds and van der Waals interactions with the bases within the major groove the DNA helix · Some proteins occasional interact with the sugar phosphate backbone and with the minor groove · Many binding motifs and domains are found in several transcription activating factors o Nature finds something that works well, it makes a lot of it. DNA Binding Domain Types: 1) Homeodomain: · First described in transcription factors in drosophila · The domains are encoded by master genes that regulate the development of the fruit fly larva o These are called homeotic genes § Most important are called: Antennapedia; Ultrabithorax § Antennapedia: regulates head § Ultrabithorax: regulates thorax and abdomen · These transcription factors have a 60 AA homeodomain that is very conserved from the fruit fly all the way to the human · Motif is an alpha helix that binds to the major groove of DNA, a turn and then another alpha helix 2) Zinc Finger Proteins (different categories): · 2 categories A) C2H2 (over 1k known examples o 2 cysteines and 2 histidine and coordinate the Zn2+ ion o Usually the zinc finger protein is a monomer with 2-3 repeated c2h2 motifs § Some proteins have more o The protein motif has two B-strands and an alpha helix § Together folding to coordinate a zinc ion § the alpha helix that binds to the major groove of the enhancer or promoter proximal element. o proteins that contain these C2H2 fingers could contain several of these motifs o each motif is a finger and binds DNA with its alpha helix o In the pic we have a transcription factor with 5 C2H2 motifs and the alpha helices bind in the major groove of the DNA o a classic example of the C2H2 zinc finger protein is TFIIIA (Transcription Factor for RNA pol III A) § 1st TF characterized with 9 zinc fingers B) C4 zinc fingers (about 100 known) o Is found in steroid hormone receptors o Often referred to as nuclear receptors o They bind to a particular ligand for ex. Glucocorticoid and then become transcription activators. o They usually have 2 zinc fingers (C4 zinc fingers) each with 4 cysteines § Each finger binds a zinc ion o These proteins can form homodimers and heterodimers o Homodimers bind DNA sequences that are inverted with respect to their sequence o Figure shows a homodimer for zinc fingers; each binding a zinc ion shown as the grey sphere. 3) "Leucine-Zipper" proteins (e.g. McKnight's C/EBP) (a.k.a. basic zipper or bZip proteins) · Gcn4 is a transcription factor of this motif · This motif has a long extended alpha helix which interacts with another protein either identical to the first or by way of a second different protein by way of the long coil-coil interaction · The alpha helices have a hydrophobic residue every 7th amino acid in its primary sequence · When the coil is formed - the hydrophobic amino acids are projected to one side of the amphipathic alpha helix o One side of alpha helix can interact with another long extended alpha helix hydrophobic side. § 2 proteins join to form the coiled-coil · McKnight actually coined the term "leucine zipper". 4) Helix-loop-Helix proteins, a.k.a. basic HLH (bHLH) · Similar to leucine zipper o Can form dimers or homo- or heterodimers o But a loop separates the dimerization domain between two protein and the alpha helix that contacts the major groove of the promoter proximal element § 1st alpha helix contains basic AA's that bind DNA § 2nd helix has hydrophobic AA's that help form the coiled coil interactions · Ex. Leucine Formation of Homodimers and Heterodimers by: · 3 families that form these homo- heterodimers o C4 zinc finger o Leucine zippers o bHLH protiens · notice the C2H2 fingers are not included because they form monomers Do not form dimers. · Panel B - Homodimers - Target Sites 1, 2 and 3 are limited in what these proteins can bind. · But, If they can form heterodimers as shown in target sites 4,5,6 the number of promoter proximal elements these proteins can serve increases · It's called combinatorial control - mixing and matching these proteins in the family in order to activate a wide number of genes · Panel c - inhibitory factors red structures interact with an activation protein to block its ability to bind to the promoter proximal element. Cooperativity in Binding: · Demonstrated by 2 transcription factors NFAT and AP1 o On their own they bind their target DNA sequences rather weakly o If allowed to interact the complex of the 2 now binds the target sequences quite strongly o The separation of the binding sites on the DNA must be maintained for the complex to bind properly o The linker scanning assay deleted or inserted 10 bp at a time to maintain the phasing of the B form DNA-double Helix Activation Domains: · Regions of the transcription factors that bind to other proteins to activate gene expression · The other proteins are called co-activators · Help to assemble transcription initiation complexes (several proteins assembling on a promoter to initiate transcription) · Sequences of activation domains are quite diverse: o Some are acidic (rich in Glu and Asp) § Fairly unstructured until they interact with co-activators, then might form an alpha helix o Some are rich in Gln and Pro o Some are rich in Ser and Thr o Some strong activation domains not enriched with any particular AA and some are weak NOTE: various proteins form a complex to bind and interact on an enhancer element. Upon binding the proteins also interact with one another. Summary of Enhancers: · Cis-acting enhancers are the actual DNA sequences we are talking about · Trans-acting enhancers are forming the actual complex that specifically bind to DNA sequence o Called players in activating gene expression · Enhancers: o 50-100 bps in length o Bind trans-acting activators and interact with co-activators (proteins) - help stimulate gene expression o Element of DNA can lie either upstream or downstream of the promoter sequence itself that drives gene expression o Notice HMG1s: bend DNA by binding the minor groove · The enhancesome - contains high mobility group proteins, HMG1 in particular o Large body of proteins o HMG1 - A non-histone chromosomal proteins, binding to minor groove of DNA within the enhancesome to help bend the DNA § HMG1 is a transcription factor and associates with the larger complexes to help fasten form structure on the enhancer. § What are these proteins doing? How are they getting an enhancer element to stimulate gene expression?

8) When we say that the Nuclear Localization Signal (NLS) is "necessary and sufficient", we mean that when linked to a normally cytoplasmic protein, it can direct the transport of this protein into the nucleus. A) TRUE B) FALSE

A) True

5) The principle organelle shown in the majority of the image to the right is (are) A) the rER. B) the Golgi complex. C) mitochondria. D) secretory vesicles. E) plasma membranes.

A) the rER.

1) Which of the following is NOT associated with RNA Polymerase II (Pol II)? A) A long carboxyl terminal domain (CTD). B) Upstream Binding Factor (UBF). C) TFIIF. D) Mediator. E) NELF (Negative Elongation Factor).

B) Upstream Binding Factor (UBF).

CpG methylation is a mark for ___________________. From our previous Unit 3 material, which chromosome in mammals is enriched with methylated CpG's?

Besides histone marks associated with heterochromatin: · Still talking about repressing gene expression, but now we're looking at the DNA itself instead of the protein factors. o Heterochromatin - also maintained by DNA methylation · 5-methyl cytosine o Enriched in heterochromatin and involved in maintaining DNA as a silent heterochromatic form · The Xi chromosome - the DNA and histone proteins are methylated. · Recall several types of promoters are enriched with CpGs o The C's can be methylated § Silence genes o The "p" is just the phosphodiester linkage between C and G. o Methylated CpGs are marks for heterochromatin but talking about DNA · Heterochromatin replicates late during S-phase o Takes a while to open up the heterochromatin for DNA polymerase to replicate it.

What family of snoRNAs guide site-specific methylation of pre-rRNA? What family guides conversion of uridine to pseudo-uridine? (Fig. 10-42) Where do many (not all) of these snoRNAs come from?

Box C+D snoRNAs: site-specific 2'-O-methylation Box H/ACA snoRNAs: site-specific conversion of uridine to pseudouridine - most snoRNAs are derived from introns of protein genes, and all of the assembly steps are believed to occur in the nucleoplasm. - most snoRNAs are transcribed from independent genes; - few are encoded in introns (81). - Processing of the pre-snoRNAs in yeast is coupled to mRNA splicing

Chromatin Immuno-precipitations (ChIPs; Fig. 9-18)

Chromatin Immuno-Precipitations (ChIPs) · A powerful technique to identify DNA sequences associated with defined chromatin proteins fro which you have an antibody. - Localizes chromatin protein of interest in the genome. Ex, the chromatin in these living cells is treated with formaldehyde, which cross links the chromatin proteins to the genomic DNA (and of course kills the cells). - Next, the chromatin is isolated from the cells and sheared to small fragments using a sonicator. - An antibody (in this case against RNA Pol II) is used to affinity purify (immuno-precipitate) all the RNA Pol II complexes with their various cross-linked DNA fragments. - You then reverse the cross links and get rid of the chromatin proteins, and then - use Next Gen Sequencing to sequence (identify) the 1000's upon 1000's of DNA fragments that co-precipitated with all the 1000's of RNA Pol II complexes. - The sequences of the DNA fragments allow you to identify where they occur within the genome which was sequenced in its entirety years ago. - The more frequently you recover a particular DNA sequence, the higher the peak. - Keep in mind, the peaks on a ChIPs graphs represents the DNA fragments bound by RNA Pol II at the time of crosslinking.

Drosophila sex determination as determined by alternative splicing. (Fig. 10-18, 10-19)

Classic Example: Drosophila Sex Determination · A cascade of alternative splicing events in females versus males. o Produces different protein products · Starts with a gene called sex-lethal (sxl) o Protein product of sxl is an RNA binding protein with RRM § RRM = RNA Recognition Motif o In very early female embryos, sxl is expressed from an early promoter. Late promoter is silent o In very early male embryos, sxl is not expressed at all. § Neither promoter active in early male embryos Sxl activity in early and late female and male embryos: · The sex-lethal gene is active in early female embryos, but silent in early male embryos. · The early female embryos use the early promoter (PE) for this early transcription. o Male is completely repressed in early genesis · The Late promoter (PL) is silent in both early female and male embryos. · The early female pre-mRNA will splice its two exons to produce an early sex-lethal protein in the early female embryo · Note that exon 2 in the early female embryo encodes an RNA Recognition Motif (RRM). So the sex-lethal protein in early female embryos is an RNA-binding protein. The early male embryo has no sex-lethal protein product. · In later embryogenesis, both female and male embryos use the late promoter (PL) on the sex-lethal gene. · Both late female and male embryos produce the same pre-mRNA, but only the late female embryo contains the early sex-lethal protein which binds directly in front of exon 3 of the late pre-mRNA to block its inclusion into the mature mRNA. · Note that exon 4 in the late pre-mRNA is the same as exon 2 in the early mRNA. Therefore, the late sex-lethal protein in late female embryos is again an RNA-binding protein. · The late male embryo undergoes what is called "default splicing" - in other word, there are no protein factors regulating splicing. Note well that exon 3 is now included in the late male's mature mRNA. This exon has a STOP codon in frame with upstream codons encoding the sex-lethal protein. The result is, exon 4 that encodes the RRM is never translated in male embryos. So the truncated sex-lethal protein in late males is non-functional. o Nonfunctional because you do not translate past exon 3 · In females only exon 1, 2 and 4 are used. Functional sxl protein produced · The sxl protein missing in male and undergoes default splicing · Exon 1, 2, 3 and 4 are included in mature mRNA · Exon 2 is the same in early and late embryogenesis Summary of important events of sxl activity in early and late embryogenesis of males and females: · Late promoter is active in both males and females o later in embryogenesis. o late promoter activity continues throughout development. o early Sxl protein in females ensures female-specific splice patterns for late sxl pre-mRNA. § i.e. female splice pattern prevents use of exon 3. · Note: exon 2 in early female protein = exon 4 in late female protein. o This exon encodes an RNA-binding domain (RRM). o without early Sxl protein in males: § males have a "default" splice pattern. § males produce a non-functional Sxl protein. · due to presence of exon 3 and its stop codon · i.e. exon 4 never expressed in male protein. · therefore, no RRM Late female Sxl protein now regulates splicing Transformer (Tra) pre-mRNA, but again only in females: · Other genes involved in sex determination involved in Drosophila · Sxl shown on top and it's the late pre-mRNA found in males and females · The late sex-lethal protein is functional only in female embryos. It feeds back on its own sex-lethal pre-mRNAs to ensure that exon 3 is excluded from the mature mRNA in females. · Male embryos contain the non-functional sex-lethal protein, so again, exon 3 is included by default splicing in the mature sex-lethal mRNA. o Includes exons 2, 3, 4 in mature mRNA · The red bar represents the STOP codon. o So, the male embryo never makes a functional sex-lethal protein. · The functional late sex-lethal protein in females also works on the transformer (tra) pre-mRNA. · The story is similar: the sex-lethal protein block in inclusion of exon 2 in the mature tra mRNA in females. · The tra pre-mRNA in males again undergoes default splicing to include exon 2. · Exon 2 has a STOP codon in frame with the codons encoding the tra protein. o So, no functional tra protein is made in males. · The tra protein does not have an RRM, but it interacts with transformer2 (Tra2) that does have an RRM. · In female embryos, tra interacts with tra2 and Rpb1 to bind exon 4 in the double-sex pre-mRNA, but only in females. o This ensures that exon 4 is included in the female double-sex mature mRNA. o Exon 4 has a polyA signal sequence directing cleavage and poly-adenylation. · In late male embryos, there's no functional tra protein, so nothing binds exon 4 of the male double-sex pre-mRNA. o So, in males, exon 4 is spliced out as a larger exon, linking exon 3 to exon 5. · The end result is: both female and male embryos produce double-sex proteins, but they are very different form each other. Rbp1 and Tra2bind to six sites in exon 4 but only if Tra is present; assures female-specific splice choice. · female embryos the transformer yellow protein binds to the Rbp1 and Tra2 complex o Tra2 is the RNA binding protein · The triple complex binds to Exonic Splicing Enhancer sequences (ESEs) o In exon 4 o Only in female embryo where a function Tra protein is produced · The male does not have the Tra protein so it undergoes an alternative splice choice linking exon 3 and exon 5 · Because the Tra Rbp1 Tra 2 complex binds to these ESEs in exon 4 of female pre-mRNA, forces splicing to include exon 4 o Exon 4 has a Poly-Adenylation sequence stimulates the cleavage and Poly adenylation of exon 4 o In female, the mRNA ends in exon 4 · In male, exon3 is ligated to exon5 and then ligated to exon6 which has a poly-adenylation signal stimulating cleavage and poly-adenylation · Notes: In late female embryos, the Tra-Tra2-Rbp1 protein complexes bind Exonic Splicing Enhancers (ESE's), ensuring that exon 4 is included in the female's mature double-sex mRNA. · Males don't have this complex, so exon 4 is skipped over, and exon 3 is ligated to exon 5. Summary of Important points: · Tra protein does not have a RRM, but it interacts with Tra2 that does. · Tra-Tra2-Rbp1 complex controls female specific splice events for o double-sex pre-mRNA. o exons 3 and 4 expressed in female Dsx protein. o Female Dsx protein represses genes necessary for male development. Conversely: · Male dsx pre-mRNA undergoes a default splice. o exons 3, 5 and 6 are used, but not exon 4. o Male Dsx protein represses genes necessary for female development. · The sxl protein functional in females directs splicing of Tra pre-mRNA in females · The functional tra protein in females then influences the alternative splice choices of the mRNA · The male undergoes a default splicing mechanism because it lacks the sxl protein and the tra protein · Many examples of alternative splicing to give rise to different protein isoforms from the same gene. · Alternative splicing is mediated by certain auxiliary RNA-binding proteins (splicing factors): o Examples: o Factors with SR motifs may be differentially expressed. o Relative concentrations of certain other RNA-binding proteins, like hnRNP A1 - influence alternative splice choices with its abundance in certain cell types § hnRNP proteins - the alphabet proteins

Primer extension assays to measure RNA abundance.

Control (wild type) promoter produces shorter RNA (due to 10 bp deletion) Primer extension assay: - Reverse transcribe RNA using a promoter, reverse transcriptase and 32P-dNTPs - Each linker scanning mutation had to tested for transcription efficiency using Primer Extension Technique. - The mutant promoter is shown on top, and this promoter drives transcription of a reported gene - To determine how much RNA is transcribed off this mutant promoter, an oligonucleotide (primer) anneals to a region 90 nts downstream of the 5'end. - Reverse transcription in the presence of radioactive dNTPs extends the primer (Primer Extension) to synthesize a short cDNA. The more RNA tat's present, the more cDNA made..

4) Which of the following sorting signals is required for the transport of BiP back to the rER? A) Stop-transfer anchor (STA) B) Signal anchor (SA). C) Amino terminal signal sequence. D) KDEL E) Mannose-6-Phosphate.

D) KDEL

3) RNA Polymerase III (Pol III) transcribes all of the following genes EXCEPT A) tRNA genes. B) 5S ribosomal genes in metazoans. C) 5S ribosomal genes in yeast. D) The gene encoding the U1 snRNA. E) The gene encoding the U6 snRNA.

D) The gene encoding the U1 snRNA.

Demonstrating specific interactions between transcription factors and their cognate DNA elements: DNase I foot printing (Fig. 9-24), mobility shift assays (Fig. 9-25), in vivo transcription assays (Fig. 9-26).

DNase I foot printing: · Once you purify the protein factor test it with DNase I foot printing · Once you have your transcription factor (protein) purified, you have to show that it specifically binds to the enhancer or promoter proximal DNA element. The DNase I foot printing assay can show this. The enhancer DNA element is end-labeled with 32P, and Dnase I is added in very low concentrations so that on average it cuts one DNA molecule in the short time you allow for the reaction. On the left side of the figure, multiple DNA fragments are cut by Dnase I at the arrows. When you run the DNA fragments out on a polyacrylamide gel, you see only the radioactive fragments by autoradiography. Notice that without the protein factor DNase I can cut the enhancer element in green, but the protein factor blocks DNase I from cutting the enhancer element. DNase can cut in front and behind the protein factor, but not where the protein is bound to the enhancer element. You then separate the cut fragment on a polyacrylamide gel to see the foot print (next slide). · David Galas invented DNase Foot Printing. · Would also want to run a mobility shift assay. · Where your protein bound represents the "footprint" (the red box). Bands below the foot print result from DNase cutting to the left of your protein on the previous slide, and bands above the foot print are sites to the right of your protein where DNAse cut (these are larger fragments end-labeled on the left side). But your protein protected the enhancer DNA sequence from being cut, so no bands appear in the footprint itself. · This is one of McKnight's footprints. The guy who inverted Dnase I foot printing is David Galas. I have a funny story about my interviewing at USC in LA where Galas was Chairman of the Biology Department there (ask if you're interested). Mobility Shift Assay: protein binds the labeled DNA fragment to shift it upwards in electrophoresis · A second assay that can show specific binding between your transcription factor and the DNA enhancer or promoter proximal element is called Mobility Shift Assay. Here the DNA is the radioactive probe (end labeled wit 32P). · When your protein factor binds the DNA, the DNA-protein complex now has greater mass in the gel system, and the complex shifted upward in terms of slower mobility in the gel. Most researchers would run both DNase foot printing and Mobility Shift Assays to confirm that their protein is binding the DNA enhancer element in a sequence-specific manner. In vivo transcription assay: · Once you have the trans-acting protein (e.g. C/EBP) purified: o Edman degradation to get partial AA sequences. o Then reverse genetics to get the gene. o Clone the gene (call it gene X). o Test it by transfection and in vivo transcription assays.

What's the main pathway for mRNA degradation in the cytoplasm? (Fig. 10-27a). What two other pathways are known? (Fig. 10-27b, c)? What are Decapping Proteins 1 and 2, Xrn1, and the Exosome doing? What are P bodies? What marker proteins are found in P bodies?

De-adenylation-dependent decay - figure - Most prominent mechanism of mRNA turnover in a eukaryotic cell Others: De-adenylation-independent decay Endonuclease-mediated decay - DC proteins 1 and 2 remove the 7-methyl G cap - XRN1 chops away at 5' end of mRNA - Exosome chops away at the 3' end of the mRNA - P bodies are sites of translational repression and mRNA degradation. [Dynamic: grow and shrink.]

Linker scanning mutations (Fig. 9-22): what are we doing here? Why are the linkers 10 bps in length?

Determine what promoter regions are important for efficient transcription. Why 10 bps? - The linkers are 10 bps in length because each turn of the B-form double helix is 10 base pairs. - Don't want to change the phasing of the major groove because this is where most transcription factors bind. You're changing the sequence, but not the major groove phase with respect to sequences upstream and downstream of the insert.

2) Which of the following techniques would be BEST to verify that your favorite transcription factor specifically binds a particular promoter-proximal DNA sequence? A) R-looping. B) Phase contrast microscopy. C) Primer extension. D) Yeast two-hybrid. E) DNase I foot printing.

E) DNase I foot printing.

7) The technique used to display Dynamin in the side picture is A) Immuno-fluorescence microscopy. B) Immuno-precipitation. C) Fluorescence Recovery After Photo-bleaching (FRAP). D) Photo-Activated Localization Microscopy (PALM). E) Immuno-electron microscopy.

E) Immuno-electron microscopy.

6) Nuclear import of a 100 megaDa protein requires all of the following EXCEPT A) a Nuclear Localization Signal (NLS). B) Importins alpha and beta. C) the nuclear pore complex. D) Ran-GDP. E) the cap-binding complex.

E) the cap-binding complex. 1) Importin α binds the NLS on Cargo 2) Importin ß binds α 3) In the nucleus, GDP is swapped out for GTP;RCC1 is the exchange factor (chromatin protein) 4) Once inside, Importin ß binds Ran-GTP 5) Cargo is released. 6) α and ß return to the cytoplasm coupled toRan-GTP 7) Ran-GTP is converted to Ran-GDP b yhydrolysis of GTP. Bipartite NLS

Energy requirements for ER translocation (lecture slide).

Energy requirements for Translocation · Recall that P54 of SRP and the a subunit of SRP receptor are guanine nucleotide-binding proteins (switch proteins). · The far left shows the SRP receptor, a subunit has GTP bound and interacts with the translocon which is closed at this point. · As the ribosome, with its nascent secretory protein and signal sequence arrives to this translocon delivered by SRP and its GTP bound configuration o The SRP interacts with the SRP receptor o Both have GTP bound · Ribosome delivered to translocon, gate is open and co-translocation insertion of the secretory protein occurs · Both the SRP and the SRP receptor hydrolyzes its GTP allowing dissociation of the SRP from the SRP receptor o Job done o LSU sits on top the translocon and the ribosome continues with co-translational insertion of the protein into the ER lumen

Eukaryotic RNA polymerases versus prokaryotic RNA polymerase (Table 9-2; Figs. 9-12 and 9-13) Don't forget alpha aminitin.

Eukaryotes: - 30 nm fiber is silent genes maintained by transcription repressors. - pioneer proteins open up the 30nm fiber and other transcription co-activators will bind to recruit RNA Polymerase. The presence of these factors determine the rates of transcription. RNA Pol II has a Carboxy Terminal Domain (CTD) - has more subunits - located in nucleoplasm - (5'7mG cap); mRNA; snRNAs; siRNAs; miRNAs RNA Pol I : - has no CTD - located in nucleolus - Transcribes pre-rRNA (28S, 18S, 5.8S rRNAs) - very sensitive to Actinomycin D at low concentrations - low sensitivity to alpha amanitin - CX5461, is specific for Pol I RNA Pol III - do not have a CTD - located in nucleoplasm - tRNAs; 5S rRNA; Sn RNA U6; 7S RNA; Other small stable RNAs - very sensitive to alpha amanitin - intermediate sensitivity to alpha amanitin Bacteria: RNA Pol is made of 5 subunits - beta, beta', omega, alpha I and alpha II - one RNA Pol transcribes all RNA in the cell - no CTD Yeast has structural similarity to bacteria RNA Pol Eukaryotes Pol I and III have alpha and omega like subunits (similar to bacterial alpha and omega subunits). Note CTD (carboxy terminal domain) on RPβ1 for only Pol II. a-like subunits for Pol II are different than the other alpha subunits in eukaryotes. Other smaller subunits are common in all three eukaryotic polymerase complexes.

Trans activator proteins: DNA-binding and activation domains. (Fig. 9-27, 9-28)

Eukaryotic Transcription Activators and Repressors (proteins) 2) Genetic identification and characterizations: (this approach was used in yeast) · GAL4 in yeast is the wild type gene that encodes the transcription factor, Gal4 o Gene identified by italics and all capital letters · gal4 is a recessive mutation in the gene o identified by lowercase italicized letters · UAS = Upstream Activating Sequence = promoter proximal regulatory sequence/element · UASGAL4 is a 17 bp sequence that Gal4 binds to specifically o Gal4 binds to activate transcription in WT cells grown in galactose, but not in cells that are gal4 (producing a mutant Gal4 protein). o UAS is upstream of transcription start site. NOTE: Yeast was used heavily to identify transcription activators and repressors. Gal4 protein has two functional domains: DNA-binding and Activation · Gal4 is a transcription factor o Wanted to dissect its amino acid sequence and functional domains o They deleted portions of cDNA perhaps encoding the amino or carboxy terminus or perhaps the middle of the protein by deleting regions of the DNA encoding these Domains of the protein · The purple arrows. They deleted cDNA that encodes the first 50 aa. · B-galactosidase activity drops to 0. The first 50 aa of Gal4 are important to initiate transcription · Further down you can see more and more portions of the carboxy terminus end are deleted. o B-galactosidase activity decreases o The back end of protein is also important for initiating transcription of the reporter-B galactosidase activity · The internal region was deleted, leaving amino terminal end intact and the carboxyl end of protein intact. o They got full B-galactosidase activity o The central region of protein is not important for transcription initiation · Amino terminus Domain encodes a DNA-binding domain · Carboxy-terminus encodes an Activation domain Other transcription factors show similar modular constructions · There are many other transcription factors: o Gal4 § We know this one has a DNA-binding domain at amino terminal end and an activation domain at the carboxy end o Gcn4 § DNA-binding domain at the carboxy end § Activation domain at the central region. · We can mix and match these domains for designed purposes of regulating the expression of cloned genes.

What are translationally dormant mRNAs? And how do they become active for translation (Fig. 10- 31).

Example 2: Polyadenylation related to translation initiation · Sequences in RNAs that can Regulate stability or expression is the CPE, shortly in front of the Poly(A) signal sequence · mRNAs found in e.g. oocytes, eggs, or very early embryos · Maternal RNAs that remain silent until needed · Very short poly(A) tail · CPE = cytoplasmic polyadenylation element · CPEB = binding protein with an RRM and zinc finger · The maternal RNAs have the CPE elements and a protein called CPEB that binds to the element · CPEB has an RNA recognition motif and a zinc finger o Zinc fingers can also bind RNA · CPED interacts with maskin · Maskin then binds to eukaryotic initiation factor 4E protein which normally binds to the 7-methyl G cap on mRNA · Maskin prevents the other initiation 4 factors from assembling with 4E o No translation initiation when maskin is binding the 4E protein · The maternal message held for weeks maybe months has a very short poly(A) tail o Something nipping away and making it short but the RNA is not degraded away § Very important and needs to be expressed at some point in embryogenesis · during frog oocyte maturation, CPEB is phosphorylated which releases Maskin · Cytoplasmic versions of polyadenylation factors then extend the poly(A) tail · PABPI then binds the poly(A) tail and stabilizes translation factors. o Similar mechanisms in neurons, perhaps for learning and memory

What are iron response elements, and how do they regulate translation of the transferrin receptor mRNA (Fig. 10-33b).

Example 3 of Normal regulation: · Transferrin is a vertebrate serum protein that carries iron. · Transferrin receptor (TfR) in PM binds transferrin to deliver iron into cells if they require it · TfR mRNA is regulated: iron response elements (IREs) in the 3' UTR and with the IRE-Binding Protein. · Transferrin is a protein and cannot cross plasma membrane on its own to deliver iron o A transferrin receptor protein that binds transferrin to deliver it and its iron into the cell · The transferrin mRNA is regulated by sequences in the 3' untranslated region of the mRNA o The sequences are called the iron response elements (IREs) § They bind a specific protein the IRE binding protein · If the cell has plenty iron and doesn't need to acquire more the transferrin receptor is very unstable o The IREs form stem loop structures o Signals the mRNA is short lived and is degraded very quickly § Cell doesn't need iron so doesn't need to express the transferrin receptor o The mRNA has a short half-life from IRE not binding the IRE binding protein · If the cell requires iron it needs to produce the transferrin receptor o Here, the IRE binding protein binds to the stem loop structures, the IREs and prevent the degradation of the mRNA encoding the receptor o Receptor is made and goes to plasma membrane and binds transferrin § Delivers transferrin with its bound RNA to the cytoplasm · Like a thermostat · Many examples of regulated mRNA stability. What happens when things are not normal? · Part of gene expression is to also get rid of mRNA

T/F. GPI-anchored peripheral membrane proteins interact with the cytoskeleton.

False

T/F. In general, splicing needs to be completed prior to export. (See Fig. 10-26 as it describes how HIV circumvents the rules in an infected cell.)

False, HIV Rev protein: - Only completely spliced mRNAs are exported through the NPC into the cytoplasm - HIV is a smart virus and gets around that requirement of completing splicing before exporting to cytoplasm - In early transcription of HIV, mRNA they will undergo splicing and will be exported to the cytoplasm - The mature RNAs from HIV will encode the REV protein - Rev protein migrates back into the nucleus of the cell and bind to the Rev Response element - transcribed into HIV pre-mRNA that will not undergo complete splicing - Rev protein will facilitate the export of these HIV mRNA from the nucleus into the cytoplasm without the mRNA having been completely spliced - The HIV virus is exporting mature mRNAs that do undergo splicing and with rev protein present pre-mRNA that have not completely undergone splicing are also exported. 9kb = 4kb = 2kb are exported into cytoplasm HIV can encode different proteins Bypassing the requirement - of splicing needing to be completed to be exported to cytoplasm.

General Pol II transcription complex assembly (Fig. 9-19; but I like the lecture slides better).

Formation of the RNA Polymerase II pre-transcription initiation complex 1. TFIID is a large complex of 14 proteins, one of which is the TATA-binding protein. So TFIID is the first complex to bind the promoter at the TATA box if it's there. 2. TFIIA then also binds, in part binding TBP and DNA. TBP is a monomer, but its two halves show 2-fold symmetry. Recall that TBP binds the minor groove to bend the TATA box quite severely, ultimately to force the two DNA strands apart for transcription. HMG1 (High Mobility Group 1) proteins also bind the minor groove. 3. TFIIA and TFIIB join. TFIIA is a hetero-dimer and TFIIB is a monomer. TFIIB binds within the major groove of upstream BRE - TFIIB binds to the TFIIB Recognition Element (BRE) just upstream of the TATA box. 4. A preformed complex of RNA Pol II and TFIIF joins (tetramer (a2b2). - TFIIF delivers the Pol II complex. 5. TFIIE joins. (tetramer (a2b2). - TFIIE creates a docking site for TFIIH 6. TFIIH consists of 9 protein subunits. - One subunit has helicase activity (Ssl2) to create an open DNA complex using TP hydrolysis. - Some TFIIH subunits, including the helicase are also required for DNA repair. - TFIIH was used in Nucleotide Excision Repair with several of the XP (Xeroderma pigmentosum) factors Order in which the General Transcription factors load onto the promoter. o D → A/B → F* → E → H o * Brings in RNA Polymerase II

hnRNP proteins: who are they? What do they do? (page 350).

Heterogenous Nuclear Ribonucleoprotein Particles (hnRNPs): · Contain heterogenous nuclear RNA (aka, pre-mRNA) · Proteins coat pre-mRNAs to protect and stabilize RNA structures · Several hnRNP proteins have been identified: o Named by the alphabet: § A1, A2, B1, B2, C1, C2, D, E,.....U · A1 and A2 are splice variants from the same gene. o Antibodies prepared: localization and immune-precipitations o Their cDNAs obtained by expression cloning, were sequenced: § Deduced amino acid sequences revealed common peptide motifs associated with RNA: · RRM motif (a.k. RNP or RBD) · RGG motif (Arg-Gly-Gly) · KH domain (found in K protein in alphabetical list above) · SR motifs (Ser-Arg) - usually are involved in splicing Function of hnRNP Proteins: (A-U for nascent RNAs) · Maybe they act as RNA chaperones, keeping nascent RNA from mis-folding, or perhaps stabilizing correct folding. · Without hnRNP proteins, nascent RNA could form dis-advantagious stem and loop structures inhibiting function · With hnRNP proteins, keep RNA from misfolding to interact with many other molecules.

What are hydropathy plots and what are they good for (Fig. 13-16)?

Hydropathy profiles: · Allows you to predict Topogenic sequences o Regions rich in hydrophobic of amino acids appear as peaks above 0 · Taking the linear amino acid sequence of a protein and asking a computer to identify the hydrophobic regions of the protein. · At the beginning of the protein in the amino terminus we can find a sequence probably like the signal sequence and later down finding the anchor sequence which are hydrophobic regions about 25 AAs in length that show as hydrophobic pockets in the protein o Might serve as the transmembrane helices o But we know they are either signal sequence or stop transfer signal sequence.

Mediator (Figs. old 7th ed. 7-43, 8th ed. 9-39, 9-40).

Many protein subunits make up a Mediator: · Bring in general transcription factors. · The mediator in S. cerevisiae (budding yeast) and humans are very similar in structure. Cooperativity in protein binding and complex assembly: · HNF3 and HNF1 that are expressed only in hepatocytes · These activators with CEBP, HNF3 and AP1 are going to bind to the enhancer element some distance upstream of the promoter proximal region and the TTR gene itself. · We have the general transcription factors TFIID, B, H, F, E · We have a mediator, a very large protein complex, · In the assembly of the large initiation complex. The proteins that bind enhancer element about 1.7 kb upstream of the promoter proximal region and have bound their enhancer element. Their activation domains interact with the general transcription factors and with mediator to bring about the assembly of this large proteinaceous complex all to bring in RNA Pol II to get transcription initiated on this TTR gene o Expressed only in hepatocytes because of the specificity in expressing HNF1 and HNF3.

Chromatin Remodeling Complexes: SWI/SNF as a chromatin remodeler. What does it do, and how is it different from HATS?

Introducing Chromatin Remodeling Complexes: · Swi/Snf are unique to yeast (several known chromatin remodeling complexes known in higher eukaryotes) · Homologous to known helicases. o Helicases - peel apart dsDNA but remodeling complexes separate the two strands of DNA using ATP hydrolysis. · Remodeling complexes open chromatin structure but, without acetylating the core histone proteins · The remodeling complexes are changing the conformation of the nucleosome core and the DNA wrapped around it. o Remodeling the structure of: § Either changing the core histone proteins § The wrapped DNA around these proteins § Or both How the SWI/SNF gene works for a particular yeast Gene: 1. Activator SWI5 binds to an enhancer and recruits SWI/SNF remodeling complex in blue. 2. SWI/SNF is a remodeler that opens up the chromatin by mobilizing nucleosomes. a. Changing structure of nucleosome itself or somehow how the DNA is wrapped around it. 3. An activating complex forms, (which includes Gsn5), a HAT then joins to acetylate core histones to open promoter-proximal regions. a. Opens UP the core chromatin structure 4. With the chromatin structure partially opened, another Activator SBF then binds several sites in the promoter-proximal elements within the promoter proximal region. 5. SBF then binds to several promoter proximal elements. It then binds and recruits Mediators which helps assemble the preinitiation complex made of the general transcription factors. TF II H that brings in RNA Pol II. 6. Recall General Pol II TF's: D to A/B to F to E to H

Nucleoporins have FG repeats (Fig. 13-33d). The model for nuclear export of mRNAs (un-numbered figure and Figs. 10-23 and 10-24).

Many (not all) nucleoporins have: - Rich with FG: Phenylalanine/Glycine repeats - Line the central channel of the pore (see previous slide) - gives a very hydrophobic barrier between the nucleoplasm and the cytoplasm of the NPC - Interact transiently with soluble transport proteins that escort export cargo (e.g. mRNAs) Mechanism of transporting mRNA though to the cytoplasm: - In the center, are mRNA coated with certain proteins that act as transporters or exporters are bound to mRNA and interact with nucleoporins that have the FG repeats - FG creates a very hydrophobic area in the NPC - mRNA is exported through the NPC in one direction - 5' cap is the first component to lead the way through the NPC - cap binding complex that binds to the 7 methyl G cap and is important for the transport of mRNA through the NPC - Proteins that facilitate the export of the mRNA through the NPC include an NXF1 and NXT1, transporter proteins that bind to mRNA and help escort it through the hydrophobic barrier Nuclear export of mRNAs: - Mature mRNA are exported from the nucleus into the cytoplasm - cap binding complex, is required for the export of the mRNA through the NPC - mature mRNA in the nucleus is completely coated with various export factors (ex. REF, NXT1, NXF1) - poly A binding protein at Nuclear 1 (PABN1) that binds to PolyA tail of the mRNA - mature mRNA will be exported through the NPC and some of the hnRNP proteins will help to escort the protein across the NPC (alphabet proteins from A to U) - Some of the proteins will never leave the nucleus, Others shuttle back (PolyA binding protein and N1) In cytoplasm: - different set of proteins including eukaryotic initiation factor 4E bind to the cytoplasmic mRNA and - 4E binds to the 7-methyl G cap - the poly A region will bind a poly A protein, representing the cytoplasmic version binding to the polyA tail - Only fully processed mature mRNAs are exported to cytoplasm - snRNAs U1246 are transcribed by RNA Pol II and have a 7methyl G cap

What do we mean by the "Plurifunctional Nucleolus?" Check out initial assembly of the SRP.

Many Functions within the Nucleolus: 1. Ribosome biogenesis (main function) 2. Processing of other RNAs U6 snRNA - transcribed by RNA Pol III - Transits through the nucleolus for modification tRNA processing - transit through the nucleolus for processing Telomerase RNA processing - The RNA in telomerase transits through the nucleolus by complexes 3. Initial assembly of the Signal Recognition Particle (SRP) - Thoru Pederson - Discovered SRP normally in cytoplasm in protein secretion - Assembles initially in the nucleolus 4. Stress sensor inside the cell - helps arrest the cell cycle upon stress - helps induce apoptosis (programed cell death) upon too much stress. "The Plurifunctional Nucleolus" - playing several roles (assembly of SRP, post transcriptional modifications of telomerase RNA, tRNAs and U6 snRNA, regulation of p53)

Yeast mating type loci (Fig. 9-35). Which loci are heterochromatic and which are euchromatic?

Mechanisms of Transcription Repression: · Examples of silenced DNA: 1. DNA that remains heterochromatic during interphase: a. The original definition of heterochromatin during interphase i. Could contains genes b. Ex. Now referred to as Constitutive (True) Heterochromatin at the end of this particular yeast at the ends. (HMLalpha and HMLa c. Looking at mechanism of a yeast cell can change its mating type. d. The center of the chromosome just right of centromere is an active euchromatic locus called the MAT locus e. If yeast mating type is alpha it contains the alpha gene in the MAT locus f. The alpha cell can change to an A mating cell by switching out the alpha and a DNA g. A DNA comes from the silent heterochromatic copy of the gene on the far right h. Conversely an A mating cell can change to an alpha mating cell by flipping out the A DNA for the alpha silent gene on the far-left hand side of the chromosome i. Whatever gene is flipped into the MAT locus determines the mating type of the yeast cell i. Converts a silent gene, kept near ends of chromosome, into the MAT locus by Intra-chromosomal recombination, then the gene becomes euchromatic and expressed to determine the particular mating type of that yeast cell.

How is the CpG methylation pattern inherited? (Fig. similar to that on page 405 of 8th ed.) The methylated C's lie in which groove, major or minor? Why is that significant?

Methylation Patterns are Faithfully Inherited after DNA Replication: · The cell needs to maintain the methylation pattern found on the heterochromatin DNA o An important epigenetic mechanism of maintaining heterochromatin within descendant cells like a primary stem cell in early embryos, i.e. inactive x - chromosome · DNA methylated on CpGs and both strands have methyl groups on the Cs opposite each other o Methyl groups lie within the major groove of the DNA at the sites of methylation · The old template strands still maintain their methyl groups o Daughter strands is not yet methylated o This is how the cell determines the new strand from the old template strand in case of an error from DNA Pol § Cell can correct the daughter strand o Called hemi-methylated DNA - old template strand maintains methylation and daughter strand hasn't been methylated yet · DNMT1 - enzyme recognizes hemi-methylated DNA and methylates the new daughter strand opposite the previously methylated site · The DNA also contains a CpG not methylated initially · DNMT1 recognizes only hemi-methylated DNA and not sites that were previously methylate o The sites not previously methylated are not methylated by this enzyme § Preserving methylation pattern into each cell generation.

What do we mean by transcription rates? Why is elongation an important aspect of transcription rate?

Most genes in higher eukaryotes are controlled by transcription rates Rate of transcription initiation (starts per min.) and elongation are the most important aspects in regulating gene expression. The rate of a gene's transcription is dependent on how often RNA polymerase initiates transcript and continues to elongate (move down the gene). RNA Pol II pauses shortly downstream of the transcription start site. NELF aids in this pause and binds genes downstream the transcription start site.

What are the motifs often found in RNA-binding proteins: (p. 350-351 and Fig. 8-5 in the old 7th ed; p. 422, Fig. 10-5 in new 8th ed.).

Motifs: 1. The conserved RNA Recognition Motif (RRM) domain of about 80-90 amino acids: a. Been crystallized b. Four beta strands and two alpha helices work as a unit to bind RNA c. Beta sheet forms a (+) surface that interacts with (-) RNA d. Some RNA-binding proteins contain multiple RRM domains 2. Arg-Gly-Gly (RGG) motifs: a. Several RGG repeats with interspersed aromatic amino acids (e.g. Phe) b. May bind double-stranded RNA (in stem-loop structures). c. Arg side chains are methylated. (mono- or demethylated regulating the motif interaction with RNA. 3. KH domain: a. Originally described in the hnRNP K protein b. Similar to the RRM domain, except i. Has 3 Beta strands and at least 2 alpha helices c. Binds RNA via hydrophobic interactions using one alpha helix and one beta strand d. The human fragile X gene (FMR1) encodes a RNA-binding e. Mutations in FMR1 cause mental retardation 4. SR (serine and arginine-rich) motifs a. Found in many splicing factors that control alternative splicing choices

What are NELF and DSIF doing? What's Cdk9/Cyclin T doing? (Fig. 9-21)

NELF/DSIF pauses elongation of Pol II at most metazoan promoters - In HIV infected cells, TAR stem-loop is formed before Pol II is paused and recruits Tat - Tat = anti-terminator that recruits normal Cdk9/Cyclin T Cdk9/Cyclin T then phosphorylates NELF, DSIF, and the CTD (on second residue, Ser) to relieve pause on HIV genes.

Nuclear pore complex (NPC): its structure. (Fig. 13-33a and b). How many different nucleoprorins make up that bad boy? If we called a ribosome an organelle, why not the NPC? BTW, on page 622 of the 8th ed., top of the second column, Lodish says the mass of the NPC is 60-80 kDa. Totally bogus. He probably meant megaDa.

NPCs as "organelles": ~ 125 mega Daltons. ~ 16 X larger than a ribosome. - Multiple copies of ~30 different nucleoporins - 8-fold rotational symmetry about the complex. - 8 units go into make the pore complex. Nuclear Pore Complexes: - Nuclear side has 8 filaments (~100 nm long) joined by a terminal ring. - filaments and ring form a nuclear basket. - nuclear ring is attached to the nuclear lamina. - 8 spokes radiating from the central plug. - import/export Ions, water, small metabolites, and small proteins (< ~40 kDa) diffuse freely through the pores. - Larger proteins and ribonucleoprotein particles (> ~40 kDa) (e.g. ribosome subunits) need to be actively transported. - Most RNAs synthesized in the nucleus have to be exported. (some non-coding RNAs never leave the nucleus: e.g. Xist) cytoplasmic side of NPC - Filament seen to stretch out - central transporter = Region of uncertainty - Certain nuclear porins rich in phenylalanine and Glycine, the FG nucleoporins - 8 filaments projecting into the nucleoplasm Held down by the basket - Basket is attached to the nuclear lamina Many (not all) nucleoporins have: - Rich with FG: Phenylalanine/Glycine repeats - Proteins have many of these FG repeats in their composition - Creates a hydrophobic environment - Line the central channel of the pore - gives a very hydrophobic barrier between the nucleoplasm and the cytoplasm of the NPC - Interact transiently with soluble transport proteins that escort export cargo (e.g. mRNAs)

Does mRNA export depend on Ran-GTP (as far as we know)?

Nope

Nuclear import: review the Nuclear Pore Complex (Fig. 13-33); The Nuclear Localization signals (Fig. 13-34; The Ran-GTP/GDP cycle (Figs. 13-36 and 13-37a).

Nuclear Import: · Definition: shows how proteins that work in the nucleus are directed to translocate form the cytoplasm into the nucleus. · Nuclear Localization Signal (NLS): o Found on most karyophilic proteins (karyophilic = nuclear affinity) o Karyophilic proteins synthesized in the cytoplasm, but imported into the nucleus across the nuclear envelope and through the NPC · NLS first characterized in Simian Virus 40 (SV40) Large T antigen · Large T antigen is a viral protein found in the nucleus. · How did it get into the nucleus when it was translated in the cytoplasm · Regulates SV40 genome replication and transcription. (Large T antigen wreaks havoc on normal nuclear proteins) · Mutant Large T antigens do not enter the nucleus. · mutations disrupt the NLS o Large T NLS: -Pro-Lys-Lys-Arg-Lys-Val- (zip code to get the protein into the nucleus from the cytoplasm) · The NLS in SV40 large T antigen is considered a simple NLS. The simple experiment showing the simple NLS (for the Large T antigen is Necessary and Sufficient to get the protein translocated from the cytoplasm to the nucleus. · Pyruvate kinase, a typical cytoplasmic enzyme, which normally enriches in the cytoplasm and we have an antibody against that protein labeling the cytoplasm quite intensely but leaving the nuclei unlabeled. · Fuse the NLS to a cytoplasmic protein (pyruvate kinase), and pyruvate kinase translocates from cytoplasm into the nucleus. · Simple NLS, the zip code, is necessary and sufficient to translocate the protein from the cytoplasm into the nucleus Reminder of the Nuclear pore complex (NPC) · Elaborate structure of nucleoporins that form the 8-fold rotational symmetry o 8 components making up the circular structure in the nuclear envelope. · Remember: a nuclear basket made of nuclear fibrils joined by a nuclear ring · A central transporter made of nuclear porins with FG repeats · Finally, a cytoplasmic ring, that extends cytosolic filaments into the cytoplasm · Transport occurs across the nuclear pore complexes · The karyophilic proteins (e.g. the SV40 Large T antigen) made in cytoplasm and has to transport across the NPC into the nucleus. · Conversely, there are materials made in the nucleus like mRNA that need to be exported across these NPC · 2-way traffic organelle in nuclear envelope!! Mechanism for Nuclear Import: · The cargo is for ex the Large T antigen from the SV40 virus in the infected cell · The Large T antigen has a simple NLS · There are 2 proteins and together form importin bind to NLS o 2 proteins called importin a and importin b § Importin a - directly binds the NLS § Importin b - Binds to the a protein making up the importin dimer · The importin protein is necessary to get the Cargo to translocate from the cytoplasm into the nucleus · One protein not shown is a GNSP called Ran-GDP · Ran-GDP is required for the import to occur · Ran-GDP interacts with importin b and the entire complex translocates across the nuclear pore into the nucleus · Once inside the nucleus, Importin b binds to Ran-GTP · The cargo is then released from importin alpha and goes on to do what it supposed to in the nucleus. · Bound to Ran-GTP, Importins a and b are transported back into the cytosol · Once in the cytosol Ran-GTP will hydrolyze and reestablish Ran-GDP · The cycle is of the Ran Guanine nucleotide switch protein. Simple NLS found in the Large T antigen encoded by SV40 virus: · Other NLSs have been discovered: · There's also a bipartite NLS: two short basic AAs sequences separated by ~ 10 AAs. Nucleoplasmin has one of these. o 2 AAs at one end and 10 AAs separating the first 2 from a second set of basic AAs. · Other NLSs are not as apparent (readily recognizable) as Bipartite or simple NLS. · For example: recall hnRNP protein, A1. It shuttles. (hnRNP proteins - act as RNA chaperones for mRNA, A1, A, B1, B2, C1...U) o Some hnRNP proteins like A1 exports the mRNA out of the nucleus into the cytoplasm. o A1 has to shuttle back into the nucleus using its NLS. o hnRNP A1 has a hydrophobic NLS, rather than a basic NLS o NLS in A1 overlaps with its Nuclear Export Signal (NES). § A1 has a signal to get out of the nucleus (NES) and one for import (NLS) § The NLS import overlaps with an amino acid sequence for NES, export o A1 does not interact with importin a to get back into nucleus. o Instead, A1 binds to a receptor called transportin that binds A1's hydrophobic NLS and the nucleoporins of the NPC. Another kind of NLS: · Small nuclear RNAs (snRNAs) U1, U2, U4, and U5 (Spliceosome) o Transcribed by RNA Pol II. o Therefore, they have a 5' m7G-cap. o Cap Binding Complex helps export these snRNAs to the cytoplasm (mRNAs too!!). o once in the cytoplasm, snRNAs (U1, U2, U4, U5) assemble with core proteins to form their respective snurps (U1 snurp, U2 snurp, etc.) o m7G -cap is methylated twice more in the cytoplasm § to form a tri-methyl cap (only on snRNAs not on mRNAs) o likely to block translation of snRNAs! o Tri-methyl cap is now part of a distinct NLS to get the snurps back into the nuclei. o Snurps then re-enter the nucleus for pre-mRNA splicing. § first stop is the Cajal body for nucleotide modification in the snRNAs before assembly into spliceosomes Nuclear Export Mechanism involving Ran-Guanine Nucleotide Switch Protein: Nuclear Import and Export are two sides of the same coin the coin being Ran-GDP/Ran-GTP · [RAN-GDP] is high in the cytoplasm, and is required for nuclear import. · [RAN-GTP] is high in the nucleus, and is required for nuclear export of: o Ran dependent export: o Small nuclear RNAs a protein called Exportin 1 that binds to them to export them into the cytoplasm (Exportin 1/CRM1 is the export factor) o Large and small ribosomal subunits (Exportin 1/CRM1 involved with their transport) o tRNAs (Exportin T is the export factor; see next slide) · Hey, what about mRNAs? o Recall: NXF1 and NXT1 bind indirectly to mRNA to export through the NPC. · But is mRNA export Ran-dependent? Export mechanism involving Ran-GDP and Ran-GTP · Cargo is manufactured inside the nucleus and has a NES o Could be the small ribosomal subunit or the large ribosomal subunit · Some signal on the particle will interact with exportin 1 the receptor that will bind to the cargo and then interact with Ran-GTP inside the nucleus. · Once bound with Ran-GTP the cargo will be transported out through the NPC into the cytoplasm · In the cytoplasm, Ran hydrolyzes the third phosphate on GTP o Now is bonded to GDP · The cargo is released to do its function in the cytoplasm · A protein called GAP, helps Ran speed up the hydrolysis of GTP o Accelerates dephosphorylation of the guanine nucleotide · Ran-GDP is important for import, once in the cytoplasm Ran-GDP can help import karyophilic proteins synthesized in the cytoplasm needing to get into the nucleus · Once Ran-GDP enters the nucleus there is a guanine nucleotide exchange factor o Swapping GDP for GTP, not a phosphorylation · The nucleus has a high concentration of Ran-GTP and the cytoplasm has a high concentration of Ran-GDP.

Nuclear receptors (Figs. 9-42, 9-45a, 9-43, 9-44a and b, 9-45b). Explain how retinoic acid can function as a HAT and a HDAC.

Other Protein Activators Respond to: 1. Lipid (membrane) soluble hormones (e.g. hydrophobic) a. Hormones that will bind to proteins inside the cell and these proteins will act as transcription activators b. Cortisol, Retinoic Acid and Thyroxine are fairly hydrophobic - pass across plasma membrane and bind their cognate receptor proteins which then activates transcription. · Nuclear Receptor Super-Family: Bind Hormones and then activate genes. o The proteins that actually bind the hormones belong to a nuclear receptor super family o They bind and then activate genes o A commonality between the genes o The variable region works as an activation region, binding other proteins. o DNA-binding Domains: in all cases the DNA binding domain are C4Zn finger (the proteins can homo- or heterodimerize) o Carboxy termini: the hormone binding domain; binds the hydrophobic hormones (lipid) o GR = Glucocorticoid receptor An in vivo functional assay using fusion proteins · Determines how the nuclear receptors are actually working. · Panel A we are looking at mammalian cells grown in culture o A negative control o Expressing e. coli beta galactosidase § A reporter protein for us § An antibody detects the protein when expressed in the cells o Beta galactosidase remains in the cytoplasm whether or not the hormone dexamethasone (Dex) a glucocorticoid is present § Looking at glucocorticoid receptor as it binds this hormone o Not expressing the glucocorticoid receptor so B galactosidase remains in the cytoplasm plus or minus Dex · Panel B - B-galactosidase is fused to the glucocorticoid receptor with its activation domain, its DNA binding domain, and its hormone binding domain in that order. o Without Dex, the fusion protein remains in the cytoplasm § Add Dex, the fusion protein, translocates from the cytoplasm to the nuclei · What you need to get the cell to translocate is the hormone itself. o Receptor binds hormone and translocates into nucleus · Panel C - eliminate the activation domain and the DNA binding domain. Only the Hormone binding domain is fused to B gal. o In presence of Dex, the fusion protein translocates into nucleus o Without Dex, the fusion protein stays in the cytoplasm. · Slide says, the hormone binds to the ligand binding domain of the glucocorticoid receptor. This allows the receptor to move inside the nucleus. Once inside the full hormone receptor acts as a transcription factor using its DNA binding to domain to bind to target DNA elements. DNA Elements recognized by nuclear receptors: · All nuclear receptors contain C4Zn finger elements Inverted Repeats: A. looks at glucocorticoid receptor and the element of DNA it binds. a. The DNA element is an inverted repeat of itself so the glucocorticoid receptor dimerizes and bind this element and each protein binding one half of the element. B. ERE (Estrogen Receptor Element) a. The DNA element is divided into two inverted repeats as seen in the GRE. Direct Repeats: C. VDRE (Vitamin D receptor element) a. The vitamin D receptor element is shown here as direct repeats not as inverted repeats D. TRE (Thyroxin Receptor Element) a. Direct repeats E. RARE (Retinoic Acid Receptor Element) a. Direct repeats · The glucocorticoid receptor forms a homodimer with opposite orientations. o The homodimers bind the inverted repeats · Conversely the nuclear receptors that bind direct repeats are heterodimers. Homodimeric Nuclear Receptors: · The glucocorticoid receptor forms a homodimer · It is first anchored in the cytoplasm by inhibitor proteins · The hormone in this case Dexomethosome, is hydrophobic. o Passes right through plasma membrane · It then binds to the hormone binding domain, the same as the ligand binding domain in the figure. · Once bound to the LBD, it changes confirmation and is released by the inhibitors. · The glucocorticoid receptor now with its bound hormone, then translocates out of the cytoplasm into the nucleus. o Remember it forms a homodimer to bind the inverted repeats found in the glucocorticoid receptor element - a promoter proximal element for certain genes. o The DNA binding domains are binding to that element and the activation domains are present to recruit other co-activators.

What's the difference between PABII and PABI? (See lecture slide for Fig. 10-15).

PABII - coats the nascent RNA in the nucleus since there is no naked RNA in the cell. PAB1 - mRNA in cytoplasm then poly A binding I will decorate the Poly(A) tail.

RNA-Editing: What is a kinetoplast and where do you find it? How do guide RNAs edit the kinetoplasts pre-mRNAs (special lecture slide and pp 364-365 in the 7th edition, pp 439-440 in the new 8th ed.). How prevalent is RNA editing in mammals? We talked about one example (Fig. 10- 22).

Post-Transcriptional Control of Gene Expression: RNA Editing: - Mature mRNA sequence is different than what the exon sequences contained in the pre-mRNA. What?!! - Occurs in mitochondria of protozoans, and in plant chloroplasts. - Kinetoplast is the one large mitochondrion in a protist cell (e.g., Trypanosoma: causes sleeping sickness). - Kinetoplast mRNAs are edited by the insertion (mostly) and deletion (less frequently) of Uridine ribonucleotides. - Editing is controlled by small guide RNAs. - Kinetoplast pre-mRNA editing progresses from 3' to 5'. - Once one region is edited, another guide RNA anneals to edit more sequences further upstream. - the guide RNA has A's and G's that direct incorporation of U's in the pre-mRNA. (Extra nucleotides) !!!!!!!!! Only 2 examples of mRNA editing known in mammals: - occurs in mitochondria. 1. C is deaminated to U by a special enzyme found in intestinal cells, but not in hepatocytes. - The gene is Apo-B gene and has several exons - Exon 26 is one we looking at - In hepatocytes the codon we are looking at is -CAA- found in exon 26 - Translation will produce a full-length protein called Apo-B100 (LDL cholesterol) - The same pre-mRNA in intestinal cells undergoes editing (-CAA- becomes a -UAA- by deamination of C to give the U) [-UAA- is a stop codon] - Both Apo-B100 and Apo-B48 carry serum (LDL-cholesterol). - only Apo-B100 binds LDL-receptor on cell surfaces, while Apo-B48 does not.

Pre-mRNA processing in eukaryotes includes a) _______________ b) _________________ c)_______________________ (Fig. 10-2).

Pre-mRNA in eukaryotes: Processing Includes: 1. 5' capping 2. Splicing 3. 3' End Cleavage and Polyadenylation

Pre-ribosomal RNA processing (Fig. 10-40. Fig. 8-38 is far too complex, so go with the one provided in lecture describing cleavage reactions). The point is: several cleave reactions to generate 18S, 5.8S, and 28S mature rRNAs.

Pre-ribosomal RNA Processing in the Nucleolus: - Recall: Ribosomal RNA Genes are Repeated in Tandem. - Each gene is often referred to as a Transcription Unit (TU). - Overall structure of the pre-rRNA genes is well conserved. - Humans at 5' end is the external transcribed spacer, in front of the region containing the 18S ribosomal RNA - Just after is another internal transcribed spacer before the 5.8S and same for 28S and transcription starts shortly there after - 5S rRNA is transcribed by RNA Pol III Pre-rRNA processing includes: 1) Cleavage reactions - cut out 18S, 5.8S and 28S regions as they assemble 2) Base methylations 3) Pseudo-uridine conversions from Uridine 4) 2'-O-methylation of what will be the mature rRNAs. 5) Assembly with ribosomal proteins from cytoplasm and the proteins assemble with ribosomal subunits - Site- (nucleotide) specific modifications are guided by small nucleolar RNAs (snoRNAs = guide RNAs) - Direct enzymes to particular nucleotides in pre-rRNA Processing of pre-rRNA includes endo-nucleolytic cleavages and trimming to cut out the mature ribosomal RNAs.

What three kinds of eukaryotic promoters did we describe in lecture? What's a TATA box? What chemical modification can occur on CpG's?

Promoters: 1. TATA Box promoters - TBP - TATA sequence is -26 to-31 pairs upstream at the +1 transcription start site. Directs Pol II. - TFIIB (Pol II recruiter) binding site is just upstream the TATA sequence. - transcription begins with the initiator element at +1. - Downstream core promoter element (DPE) - helps TFIID bind to the promoters that lack TATA box. 2. Initiator elements: - no TATA box 3. CpG- rich promoters: - No TATA box - for genes encoding enzymes for intermediary metabolism "house keeping genes" - initiate transcription in a 20-200 bp "broad" region. - multiple transcription start sites = multiple 5' ends. - C's can be methylated (gene silencing)

What's R-looping. Fig. 10-6b in the text book really sucks. Go with the lecture slide.

R-Looping (Splicing): · RNA-looping: an early (late 1970's) indication of eukaryotic pre-mRNA splicing · B-globin Pre-mRNA with intron annealed back to its gene · Mature B-globin mRNA annealed back to its gene. Without intron in the mRNA, intervening DNA remains double stranded.

Mechanisms of repression. (Fig. 9-37a, and Figs. from lecture)

Repressor proteins: · Assist in shutting down gene expression · Block cis-acting DNA elements - promoter proximal elements o Similar to an activator that activates the gene by binding similar elements · Knockout of these repressor proteins leads to constitutive gene expression o Could be bad · Looking at a proto-oncogene called EGR-1 o Needs to be tightly controlled by repressor proteins o One is encoded by the WT1 - Wilms tumor gene § Loss of gene function leads to kidney cancer · EGR-1 is overexpressed § Protein product WT1 is a gene repressor. · Like activators it has DNA-binding domain but now has repression domains with basic or hydrophobic amino acids to interact with other co-repressor type proteins. How can individual genes be repressed? 1. DNA that remains heterochromatin during interphase: Constitutive 2. Silencing individual genes a. Facultative heterochromatin - turning off specific genes that at some time were expressed b. There are repressor proteins that have HDAC activity i. Repressor protein UME6 1. Has a DNA-binding Domain 2. Binds to an Upstream Repressor Sequence (URS) 3. Has a repression domain in red and interacts with co-repressors a. Rpd3 - a HDAC i. Takes off acetate groups on lysine amino terminus side of residues on the nucleosomes wrapping up this promoter sequence 1. Result: promoter becomes heterochromatic and silences this gene. Notes: Very similar story to activation (cooperative binding), but working in the opposite direction to now deacetylate nucleosomes on a promoter region. Repressors can inhibit by facilitating histone deacetylation as shown on the previous slide. But also, by physically blocking activators: and co-activators by binding to the cognate DNA element. a. Competitive binding with an activator. · Perhaps the repressor has a higher affinity for its DNA the activator does for its DNA. Repressors can inhibit by: b. Interaction with activation domain of bound activator. · The repressor protein binds to its specific DNA and the activator binds to its specific DNA · The repressor domain interacts with the activator's domain to prevent other proteins and co-activators from interacting with it. Repressors can inhibit by: c. Interaction with general transcription factors · The repressor proteins binds to its DNA sequence · The repressor domain then interacts and prevents general transcription factors from gaining access to the TATA box. · Other activators upstream are ineffective in recruiting other co-activators to recruit the general transcription factors all do to the repressor domain preventing that assembly to occur. · Recall the General Transcription Factors: TFIID, A&B, F, E, and H Review: Transcription factors: 1. Hyper-acetylate core histones a. (HATS) 2. Stimulate assembly of transcription initiation complexes 3. Regulate frequency of Pol II transcription starts (rates increase) a. How frequent RNA Pol II is recruited and starts transcription. 4. All by way of cooperative protein binding and complex assembly Repressors: 1. Recruit Histone deacetylases (HDACs) a. Close down transcription by forming 30 nm fiber. 2. Co-repressors: cooperative protein binding and complex assembly 3. Block transcription activators to prevent assembly of initiation complexes. Back to transcription activators: protein complex assembly · Activator proteins coming together as a protein complex to activate a particular gene in a particular cell type · For ex. Hepatocytes - TTR gene (Transthyretin gene) o Expressed only in hepatocytes due to the particular transcription factors co-expressed in this particular cell type o HNF1 AND HNF3 are hepatocyte specific transcription factors o HNF4 and C/EBP are expressed in a few other cell types o AP1 is universal and is expressed in all cell types · You need all 5 transcription factors to activate the TTR gene. · Multiple activators and some cell specific are required o Lack the specific transcription factors then the gene will be silent.

What is Rap1? What are the Sir proteins doing? What do Sir 2 and 3 bind?

RAP1 binds this telomeric DNA and recruits SIR2, 3, and 4. The SIR proteins then tend to spread forming more heterochromatin. How does that heterochromatin spread to the silent mating type genes such as HMLalpha · Still referring to the telomeric regions of chromosomes in yeast · The telomeric DNA and the protein Rap 1 specifically binds to the telomeric repeating DNA sequence · Rap 1 recruits the Sir proteins: Sir 2, 3 and 4 o Silent information regulator proteins · Sir proteins tend to spread from the telomeric region into nucleosomes further upstream from the telomer · The HMLalpha gene is to the far right and the sir proteins will silence the HMLalpha gene near the telomer Notes: Recall that telomeric DNA is constitutive heterochromatin. RAP1 binds this telomeric DNA and recruits SIR2, 3, and 4. The SIR proteins then tend to spread forming more heterochromatin. · Sir 3 and Sir 4 bind nucleosomes that are not acetylated o nucleosomes are hypoacetylated o near HML and HMR silent gene loci · if a nucleosome were to be acetylated (Right image), the Sir proteins will not bind that nucleosome. Notes: The SIR proteins bind deacetylated nucleosomes. Acetylated nucleosomes are not bound by the SIR proteins. Figure: · the telomeric region is binding Rap 1 which recruits Sir 2, 3 and 4 which spreads down the chromosome binding to hypoacetylated nucleosomes o converting the mating type yeast genes to become silent copies of these genes near the telomeric · once Sir proteins, bind to these nucleosomes, they aggregate (1 telomere, 1 chromosome) interacting with telomeres of other chromosomes. o The telomeres aggregate together in yeast nuclei. · Where do you think this aggregated telomeric heterochromatin is found inside the nucleus? Yeast cells showing telomeres that have coalesced, also co-localized with SIR 3: · Barr body's (the inactive X-chromosome which is heterochromatic) o It aggregates just on the underside of the nuclear envelope · In situ-hybridization can be used to locate the telomeric DNA sequence o Can have antibody against SIR 3 o The telomeric DNA can colocalize with SIR 3 the heterochromatin binding to the DNA to convert into heterochromatin. § Spots can be referred to as nuclear domains · Find the chromatin that contains the telomeres within the domain. · Genomic DNA is stained with propidium iodide Facultative Heterochromatin: · Found on the underside of the nuclear envelope can be converted in to euchromatin. · LacI is a repressor protein from E.coli now expressed in a mammalian cell, and it binds to the Lac operator which is transgenic in these cells. o Mammalian cells express the E. coli lac repressor protein o Trans genes within one of the chromosomes that contains the lac operators. § LacI binds to lac operators which converts the DNA into facultative heterochromatin and positions just underneath nuclear envelope. · LacI-VP16 is a fusion protein. · VP16 is an activation domain which recruits activation proteins to convert the heterochromatin into euchromatin which then disperses into the nucleus. o LacI binds to lac operator but now the activation domain VP16 recruits coactivators to open up the genes the lac operator is positioned in front of o Histone acetylase and chromatin remodeling complexes open up the condensed chromatin into euchromatin. § Chromatin repositions from underside of nuclear envelope to a more diffuse pattern through the nucleus · Good example of facultative heterochromatin converting from a condensed state to a more opened up euchromatic state.

Fig. 10-1 is a good overview of gene expression.

RNA Processing, Post-transcriptional Regulations and Nuclear-Cytoplasmic Transport · Most significant control for gene expression occurs at the level of gene transcription o i.e. the rates of transcription initiation (starts per minute) and elongation (recall pause phase) - shortly downstream transcription start site. · However, most primary transcripts in nucleus are not yet functional; true for: o Pre-RNAs need to be processed into mature RNAs. § mRNAs - capped spliced and polyadenylated § tRNAs § rRNAs · maturation occurs in the nucleus, prior to export to the cytoplasm · maturation of primary transcripts is another level (opportunity) to regulate gene expression "Gene Expression" Overview of RNA processing: · Post-transcriptional control includes all aspects of gene expression once transcription has started. o Producing a gene product. · By "Gene expression" we include all aspects of producing a gene product, whether it's a functional RNA molecule like tRNAs or ribosomal RNAs, or a protein product encoded by mRNAs. · Gene expression includes transcription and translation of course, but also the processing of the nascent RNAs inside the nucleus before they are ready to be exported to the cytoplasm. · This RNA processing, export and cytoplasmic translation of protein products is called "post-transcriptional control" of gene expression. Lamp brush Chromosomes from Newt Oocytes: · Recall radial loop model Joe Gall - chromosome guy · bright white is main axis of heterochromatin itself, stains well with DAPI · Red loops are transcriptional active looped domains. Red is an antibody that recognizes an RNA binding protein with the vast number of transcripts produced on the looped domains. o Uses RNA Pol II and RNA immediately coated by proteins. Diagram to explain one Looped Domain in the lamp brush chromosome: · RNA molecules of increasing length, three of them shown in blue arced transcription units, · Primary antibody shown in black directly binds to a particular RNA-processing protein on the nascent RNAs · Rhodamine which is a fluorescent dye coupled to the secondary antibody. The loops on the Lamp Brush Chromosomes: · They are very transcriptionally active · The RNA Pol II does the bulk of the transcription on the loops · The Pre-mRNAs are immediately coated with processing proteins o No naked RNA in the cell

Describe the properties of the signal sequence found on secretory proteins. Table provided in lecture.

Really Close Examination of: Translocation Mechanism of Secretory Proteins Across the ER Membrane: · Synthesis of secretory proteins begins in the cytoplasm on free ribosomes. · Ribosomes attached to the ER have no signal of their own to direct them to the ER. · rER ribosomes are no different than those free in the cytosol. § so, signal sequence on nascent protein is critical for getting the particular ribosome down to the ER membrane · ER signal sequence is often the first 16 - 30 AA residues at the amino terminus of the nascent protein as emerges from the ribosome. · Signal sequence directs the ribosome to the ER. · Signal sequence also directs the transport of the nascent protein across the ER membrane. · Signal sequence composition = one or more basic (positively charged) AAs, followed by several hydrophobic AAs. § First 16-30 AAs at the amino terminal end of the protien

Dave Allis made antibodies specifically against acetylated Lys #9 in Histone H3. What technique could you use with this specific antibody to determine if your gene of interest is transcribed in liver cells?

Recall ChIPs (Chromatin Immuno-precipitations): · an assay used to identify DNA sequences associated with specific chromatin proteins or histone marks o originally used to immune-precipitate RNA Pol II with an antibody to identify genes. That were currently being transcribed upon this one point upon formaldehyde fixation · this ex. Uses ChIPs used to identify nucleosomes that are modified perhaps acetylated on lysine 9 of H3. o Mark for transcription o Isolate and chromatin mechanically after formaldehyde fixation o Then add an antibody specifically for this histone mark § Maybe the acetylated lysine on histone H3 o The antibody binds to these particular nucleosomes very specifically o You can then immune-precipitate these fragments of DNA that contain the modified nucleosome o The DNA is isolated away from the protein and PCR can be used to determine whether or not your protein of interest was immune-precipitated by this antibody against this particular marl o In this case if you were looking for one particular gene you would design primers that would amplify your gene by PCR o A modern approach to this technique: § Use next-generation sequencing to identify all the fragment through the entire genome that was immune-precipitated by this antibody against this particular mark Use it to identify all the genomic fragments that were immune-precipitated

What are Cajal bodies, Histone Locus bodies, and PML bodies? (pages 468-469). BTW, who was Santiago Ramon y Cajal?

Santiago Ramon y Cajal - father of modern neuroscience Cajal Bodies: - centers for spliceosomal snRNA (snurps U1, U2, U4, U5) modification - 2'-O-methylation and pseudouridylation of these snRNAs - scaRNAs (small Cajal body associated RNAs) are related to snoRNAs (guide the 2'O methylation and the pseudouridylation in the snRNAs U1,2, 4, 5) Histone Locus Body: - Histone transcripts do not have introns and no poly(A) tail - U7 snRNA required for histone mRNA 3' processing - histone transcripts have a stem-loop. 1. cell needs the U7 snRNA 2. U1, 2, 4, 5 and 6 are for spliceosomal assembly 3. U3 in nucleolus 4. U7 dedicated to processing of histone transcripts PML Bodies (Promyelocytic Leukemia) Nuclear Bodies: - sites for the assembly and modification of protein complexes needed for DNA repair. - modification of p53; a regulator of apoptosis = programmed cell death. (P53 important transcription factor)

Compare and contrast pre-mRNA splicing with self-splicing group I and group II introns. Where do you find group II introns, and what are maturases and what encodes them? (Fig. 10-44). Where do you find group I introns? Describe Group I self-splicing introns. How is the guanine nucleotide used as a co-factor? Does this guanine nucleotide use its 2' or 3' hydroxyl?

Self-splicing of Group II Introns: - Under non-physiological, in vitro conditions, certain introns will self-splice. No energy; no protein; just RNA - Found in protein-coding genes of mitochondria and chloroplasts of plants and in mitochondria of fungi. - Mostly their pre-mRNA that undergo self-splicing These introns fold into conserved secondary (stem- loop) and tertiary structures. - Mechanism of self-splicing is analogous to spliceosomal reactions found in nuclear pre-mRNA: - branch point A nt that nu- attacks the 5' splice site leaving a OH group; It then attacks the 3' splice site in trans-esterification reactions. - Group 2 introns in mitochondria - Encode their own mRNA and in some cases, they have introns that splice themselves out (same mechanism) - removing stem loop structure 1 and 5 from the group 2 self-splicing intron diminished self-splicing - the catalyst for pre-mRNA splicing in nucleus and group 2 splicing in mitochondria is the RNA itself Maturases - Group II splicing can occur in vitro w/o proteins: - rate is very slow compared to in vivo splicing. - maturases (proteins) bind group II introns to stabilize the secondary/tertiary structures - maturases are analogous to the proteins found in nuclear snRNPs. - maturases are actually encoded by the Group II introns themselves. - are transposable elements within mitochondrial genes - Maturases also have reverse transcriptase activity. - Group II introns hop by non-viral, retro-transposon mechanism. no mutation results because.- intron spliced out Self-Splicing Group I Introns: - First characterized in the pre-rRNA of Tetrahymena thermophila. - Most organism do not have introns in pre-rRNA - The introns turn out to be a Group I self-splicing intron. - Group I introns fold into specific secondary and tertiary structures. - Introns bind a free Guanine nucleotide (a co-factor) from solution. - 3'-OH of the Guanine nucleotide is used to attack the 5' splice site. - Remember: in spliceosomal splicingin Group II and nucleus uses a 2'OH group - only one available Two trans-esterification reactions. - 3'OH group on Guanine attacks the 5' splice site, leaving a OH group - The OH group then attacks the 3' splice site. - Released Group I Intron can work as a ribozyme in vitro. Group I introns are also found in pre-rRNAs of certain mitochondria and chloroplasts. - some phage pre-mRNAs (virus infecting bacteria) - some bacterial pre-tRNAs So, what are the real enzymes involved with splicing? - The RNA molecule itself is serving as enzymes in the catalysis of splicing Tom Cech: a paradigm shift: 1989 Nobel Prize in Chemistry Shared with Sidney Altman (RNAse P)- another RNA enzyme Former BIOL 3090 & 4450 student now at U. Colorado in the RNA group.

SR proteins as splicing factors? What do U2AF and SC-35 do? (Fig. 10-13).

Serine/Arginine (SR)-rich splicing factors (4th motif) · Found in proteins often called splicing factors · Splicing factors help assemble the spliceosome · These SR-rich proteins could have one or more RNA recognition motifs o Besides SR motifs they also have RRM motifs · SR rich domains are used for protein-protein interactions. o The splicing factor proteins are helping recruit other protein factors for splicing, · Some of the SR rich splicing factor proteins, bind what are called exonic splicing enhancers (ESEs) o Part of exons · these SR rich proteins bind these ESEs · once in place the SR proteins help recruit and stabilize U1 to the 5' splice site · other SR proteins help recruit the U2AF to the back end of an intron. o U2AF is made of 2 proteins § A 65 and a. 35 kDa proteins. § The 35 kDa protein binds directly to the 3' splice site of the intron § The 65 kDa binds to the pyrimidine rich region just downstream of the branch A site. · Stabilizes U2 as it binds to Branch A site · SR proteins are splicing factors and help stabilize U1 and U2 for their splicing activities. · SC-35 - protein binds both the U2AF and the U1 snurp to help bridge the assembly of the spliceosome. o Thus SC-35 forms a bridge between the 5' and 3' ends of the intron. · By "cross-exon recognition", the authors are simply describing how these exonic splicing enhancers serve to define 5' and 3' splice sites. (acceptor and donor sites)

The SRP, the SRP receptor, and the translocon (lecture slide for SRP and Fig. 13-6).

Signal Sequence, SRP, SRP receptor and Translocon all work together in co-translational transport. · Showing the signal sequence as it emerges from ribosome in step 1. · The signal sequence then interacts with the SRP · In step 2 the SRP is interacting with the signal sequence in red as well as the large ribosomal subunit · Once the SRP has bound the large ribosomal subunit It delivers the ribosome to the surface of the ER, the membrane · In the membrane is a receptor that interacts with the SRP o Receptor consists of 2 proteins: a/b heterodimer o The a protein is also a guanine nucleotide binding or switch protein · In other words, the SRP, P54 and a protein of the receptor are both guanin nucleotide switch proteins o Interact when both of them have GTP bound shown in step 3 · Once the ribosome makes contact with the translocon channel, the GTP on the SRP and the SRP receptor are hydrolyzed to give GDP and inorganic Pi o Allows the SRP to dissociate from the receptor to be reused in another cycle · The ribosome with its nascent protein is now attached to the translocon (a large channel in the ER membrane) · The protein will be co-translationally inserted into the lumen of the rough ER o The signal sequence is then cleaved by s signal peptidase, a membrane protein in ER membrane · Co-translational insertion will continue until the protein is completely translated. o A secretory protein, completely free of the ER membrane, § Will go on to be post-translationally modified and folded The translocon: · SRP and its receptor only initiate translocation. · ribosome and its nascent protein chain are transferred to the translocon - channel for co-translational transport · -Translocon: o a set of trans-membrane proteins that form a channel through the ER membrane. o nascent protein chain continues to be synthesized. § LSU is sitting on top the translocon preventing any contact with the cytosol = a reducing environment. o passes through the translocon. o growing protein chain never exposed to the cytosol. § cytosol is a reducing environment o nascent protein chain folds within the ER lumen. § ER lumen is an oxidizing environment

Pre-mRNA splicing mechanism. (Figs. 10-7, 10-8, 10-9, 10-11).

Splicing of mRNAs: · Important regulatory sequences within the intron and at exon/intron boundaries: o 5' splice site (donor site). GU - first 2 nts of intron. 100% of the time. o 3' splice site (acceptor site) AG - last 2 nts. Foud 100% of the time. o Branch A: 20-50 nts upstream of the 3' splice site. 100% of time o Pyrimidine rich region just downstream of branch A · Length of introns can vary from 40 nts to 500,000 nts. · Yet the cell knows exactly where the 5' and 3' splice site resides. · Ligating the 2 exons together must be perfect to maintain reading frames for the ribosome in translation. Mechanism of Pre-mRNA Splicing: · Two trans-esterification reactions: o One phosphate bond is exchanged for another § "no energy required" · 1) 2'OH of branch A Nu- attacks Phosphate linkage between exon 1 and first nt of intron. Break that phosphate linkage and form one between the first base of the intron and the branch A of nt. 5' splice site (end of exon 1). o Lariat forms · 3'OH at end of first exon attacks 3' splice site (beginning of exon 2). o exons are ligated together lariat is freed · consider reading frames and possible frame shifts. Splicing Intermediates: · in vitro splicing reactions using nuclear extracts o nuclear extracts prepared from eukaryotic nuclei o we can add pre-mRNA to these extracts and ask the extracts to splice out introns to the RNAs we add o we have a nucelar extract, the globin pre-mRNA was added to it - has 2 exons and 1 intron of 130 nts - over time the invitro nuclear extract will splice out the intron. · supports the mechanistic model on previous slide · these in vitro extracts were used to identify the splicing machinery o i.e. the spliceosome - · Joan Steitz at Yale University (husband Tom Steitz) · Autoimmune disease called Systemic Lupus Erythematosus Joan Steitz Discovers Spliceosomes: · Lupus patients at Yale university medical school/hospital produced antibodies against spliceosomes. Ex. One patient with initials, S.M. · Small nuclear RNAs protein complexes (snRNAs); all transcribed by Pol II, except U6 (Pol III) 107-210 nts in length; uridine rich! o U1 - encoded by RNA Pol II so it has a G7-methyl cap o U2 - encoded by RNA Pol II so it has a G7-methyl cap o U4 - encoded by RNA Pol II so it has a G7-methyl cap o U5 - encoded by RNA Pol II so it has a G7-methyl cap o U6 - transcribed by RNA Pol III, no 7methyl G cap Note: Many small U-rich RNAs (e.g. U90-ish) in nuclei · Associate with proteins in small nuclear ribonucleoprotein particles o Referred to as "snurps" o 5 snurps § U1 snurp contains the U1 snRNA and several proteins § U2 snurp contains the U2 snRNA and several proteins § Etc. § Some proteins are common to all five snurps (core proteins) o Other proteins are unique to an individual snurp · Joan: won the Lasker award in 2018 o One of the premier RNA specialists in the world. Spliceosome function in a typical -GU---------AG-splice (an intron) 1. U1 snRNP anneals to the 5' splice site a. snRNA has stem loop structures and an SM site 2. U2 snRNP anneals to the branch point A a. Notice the branch A residue is bulged out and not part of the hybrid RNA i. It provides a 2'OH group that will nucleophilic attack the 5' splice site. 3. U4/U5/U6 snRNPs join as a trimeric complex NOTE: SM site: binds same core proteins · The SM sight on the snRNAs (purple boxes). This is the site on which the common core proteins assemble. · Lupus is an auto-immune disease, and the patient, SM, made antibodies against these core proteins. Mechanism of Snurps: · U1 anneals to 5' splice site and U2 anneals to Branch A site · Next, U4, U5 and U6 snurps join as a trimeric complex to form the intact spliceosome o In the spliceosome that the two trans-esterification reactions occur · U4 and U6 are annealed to each other before they join to make the intact spliceosome · U4 acts as a repressor RNA to keep U6 functional interactions from prematurely interacting with U2 · Once U4 leaves spliceosome, U6 interacts with U2 and it allows U2 to push the branch A nt out from the hybrid helix. · Spliceosome is complete with all five snRNPs bound to the pre-mRNA · Once intact spliceosome is formed, U4 leaves spliceosome allowing U6 RNA to reposition itself and anneal to the U2. · The U2 anneals to the branch A site to push out the Branch A nt for the first trans-esterification reaction to occur · Note the U1 snurps exits the spliceosome as well · Next, the 2 trans-esterifactio reactions will occur. · Next the hydroxyl group on the branch A nu- attacks the 5' splice site, leaving a hydroxyl group that then attacks the 3' end of the intron · U5 holds the 2 exons close together so the 2 trans-esterification reactions occurs sequentially together. · 1st transesterification reaction o The 2'Oh on branch A attacks the 5' splice site and you break that phosphate linkage as you form a phosphate linkage between the first nt of the intron (G) and branch A. o This leaves a OH at the end of the first exon. § The OH in the 2nd reaction attacks the phosphate linkage at the back of the intron · The 2nd trans-esterification rxn · The OH group left at the end of the first exon Nu- attacks the first phosphate linkage in the back end of the intron as its linked to the second exon. You break that phosphate linkage as you form that phosphate linkage between the 2 spliced exons. · The intron breaks down in most cases · U2, U5 and U6 will be recycle into another functional spliceosome. · Intron is debranched into a linear intron · Spliceosome disassembles, but individual snurps are recycled · Most not all lariat introns are degraded. · Ligated exons represent mature mRNA for translation. Recycling of Splicing Components: · From Phil Sharp's Nobel lecture on splicing · Notice: o The close juxtaposition of the two exons and U5 holds them together for the 2 trans-esterification reactions to occur quickly o No energy required for 2 trans-esterification reactions however, but ATP hydrolysis is required for spliceosome assembly and rearrangements that occur between U4, U6, and U2 o Most lariats degrade after debranching § Some introns are important and are preserved in the cell.

Fig. 14-1 is a good review.

Summarizes the mechanism of RNA export: · tRNA: o cell uses exportinT, interacts with Ran-GTP o dependent on GTP for export · CRM1/Exportin1: o Major export factor o Many things manufactured in the nucleus will interact with this factor o Exportin 1 interacts with Ran-GTP · The large and small ribosomal subunits are dependent on Crm1 · The small nuclear RNAs are also interacting with the cap binding complex and later Exportin 1 and Ran-GTP for their export · HIV rev protein binding to the rev response element o The rev protein gets unspliced mRNAs exported into the cytoplasm o By way of exportin1 and Crm1 interacting with GTP · VA RNAs: o Adenovirus RNAs and pre-miRNAs § Exported by exportin X factor · Believed to interact with Ran-GTP · Ran-GTP is important for the export of the majority of RNAs produced inside the nucleus · Not shown is the export of mRNAs, not yet linked them to Ran-GTP

T/F. Certain mRNAs are specifically localized to distinct regions of the cell.

True

Where in the cell does U3 work? (Fig. 10-43).

U3snoRNA works in the nucleolus, To carry out the cleavage reactions, cutting out (usually) the 18S rRNA

What's AU-AC splicing? And how is it different from standard splicing?

Variations on a theme: · Slightly different introns starting with AU and ending with AC instead of GU----AG · There are uique snurps that bind to these introns · Ex. U11 binds to 5' splice and U12 binds to Branch A · A special U4 and U6 that interacts with this modified spliceosome · But U5 is believed to be the standard in all. · Identical mechanism, just a rare number of introns with a different sequence and different snurps.

What are ribosomopathies? Which one do you thing dental schools are most interested in? Why?

What happens when ribosome assembly goes bad? In humans: - Several diseases/syndromes called Ribosomopathies. - Caused by mutations in genes encoding nucleolar ribosome assembly factors or the ribosomal proteins themselves. - Many affect stem cells or progenitor cells. - loss of bone marrow stem cells leads to anemia (lack of red blood cells) - loss of embryonic neural crest progenitor cells leads to craniofacial birth defects Treacher Collins Syndrome: caused by the loss of Treacle, a nucleolar ribosome assembly factor (protein

Yeast Two-Hybrid (Fig. 7-40). What is this technique doing for you?

Yeast Two-Hybrid: a technique using TFs to Determine Protein-Protein Interactions in vivo: · trying to identify other proteins that interact with your protein in vivo · done in yeast because the eukaryotic cell makes the proper post-translational modifications on the 2 proteins under investigation to determine if the 2 proteins can interact once the post-translational modifications are placed · technique relies on hybrid proteins (fusion proteins) made by ligating the cDNA to each other to produce fusion proteins o rather complicated molecular assay performed in yeast cells o first fusion protein is called the Bait Domain § the bait domain is your protein of interest · you know the protein · looking for other proteins in a living cell that can interact with your protein § It is fused with the DNA-Binding domain from the Gal4 yeast transcription activator protein · can bind a very specific promoter proximal element in yeast o the other protein is the fish domain (unknown protein) § expressed in a cDNA library where all the cDNA cloned in to the library are ligated into the cDNA encoding the activation domain from Gal4 the yeast transcription factor § this library produces a wide variety of different hybrid proteins all linked to the activation domain from Gal4 § many different FISH domains and some could interact with your bait domain Transcriptional Activation by Hybrid Proteins in Yeast: · yeast 2 hybrid assay are performed in yeast cells that are HIS- o these cells need histidine in growing medium to grow · you can transfect these cells with the plasmid that contains the WT HIS+ gene o gene driven by the UAS specifically bound by the Gal4 transcription activator protein · expression of HIS+ gene would rescue the HIS- gene in the chromosomes · can also transfect these yeast cells with a plasmid that now encode your bait protein fused with the Gal4 DNA-binding domain and a cDNA library expressing various proteins fused with the activation domain of Gal4 · notice the bait protein with its blue DNA binding domain binds to the UAS. o If the particular protein is expressed in the particular yeast cell the fish protein interacts with your protein of interest. § Restoring the Gal4 DNA-binding domain bound to your protein with the Gal4 activation domain bound to the fish unknown protein · Restoring Gal4 function § Gal4 activation domain recruits' co-activators and RNA Pol II and transcribes the HIS+ mRNA § This cell can now grow in absence of Histidine · Re-isolate plasmid that encoded fish protein and sequence through that DNA and you now know what protein can interact with your protein of interest in a eukaryotic cell after post-translational modifications.

Fig. 14-4: Classes A-E of conditional yeast sec mutations (don't memorize them, just be aware of their classification based on incremental steps in the secretion pathway).

Yeast to Genetically Define the Secretory Pathway · Discovered in Gunter bobels lab · Mutations are called sec (for secretion) mutations. Recall Sec61 subunits for translocon. o Translocon was made of Sec 61 alpha beta and gamma subunits · Temperature sensitive sec mutations. o Shifting temperature from permissive to non-permissive which blocks secretion at one of several points. o Five classes of sec mutants (A-E). o Combine different mutations to determine the secretion pathway. § Class A blocks accumulation of proteins in the rER lumen § Perhaps Sec61 alpha is mutated which blocks function of translocon itself

Describe how the eukaryotic nucleus is organized with respect to function.

discreet sites in nuclei of eukaryotic cells that conduct poly adenylation and splicing at the same time antibody against splicing factor SC-35 SR-rich protein for the spliceosome has spots where polyadenylation and splicing are rapidly occurring.

What are micro-RNAs, and how do they regulate translation of particular mRNAs? (Fig. 10-29 and Fig. 10-28). How many microRNAs have been found so far in humans? T/F. MicroRNAs are transcribed by RNA Pol III.

micro-RNAs block translation or help degrade mRNA - miRNAs encode their own genes - Transcribed by RNA Pol II - secondary structure and folds back on itself - 1900 found in humans Drosha protein: double-stranded RNA-specific endonuclease working with DGCR8 (Pasha) cleaves the pre-miRNA to ~70 nts. - pre-miRNA hairpin is processed by DICER and RISC (old friends from RNAi) - miRNAs to inhibit translation (mechanism still remains unknown) - perhaps by localizing mRNA to cytoplasmic P bodies which are sites of mRNA degradation. Summary: translation inhibition - the miRNA held by RISC forms a perfect hybrid with the 3' untranslated region of the particular mRNA - No mismatches - RISC cuts the mRNA into two fragments that are degraded by XRN1 and Exosome in cytoplasmic P bodies.

Possible orientations of transmembrane proteins (Fig. 13-10).

o Orientation is determined while they are being synthesized at the rER o Ex. Type 1 proteins, LDL receptor, has an amino terminal end in the lumen of the rER. An alpha helix in the membrane itself with the carboxyl end in the cytosol. § Lumen referred to as the exoplasmic space because of its oxidizing environment · This region becomes the outside of the cell once the secretory vesicle fuses with the plasma membrane o Type 2 membrane proteins like the transferrin receptor § Binds transferrin to deliver iron to cells § It has the opposite orientation as type 1 · The amino terminus is in the cytosol and the carboxyl end is in the lumen of the rER or the exoplasmic space. o GPCR pass through the plasma membrane 7 times - classic Hallmark for it § Carboxyl end in the cytoplasm and the amino terminal end in the lumen of the rER o GPI-linked proteins · Type I membrane proteins are usually what we deal with · How do membrane proteins reverse their orientation? o The topogenic sequences: § About 25 AAs stretches and assure the protein acquires the proper orientation within the membrane during its insertion while being translated § 2 types: · Stop Transfer Anchors (STAs) - single function · Signal Anchors (SAs) - dual function peptide sequences in the protein being translated

What is meant by co-translational transport across the ER membrane (Fig. 13-2, 13-3, 13-4)?

· (a) An experiment showing that the ER membrane must be present during the translation of the secretory protein. · Polysomes, many ribosomes translating a particular mRNA, encoding a secretory protein with a signal sequence at the amino terminus · If the ribosomes completely translate the secretory protein in the absent of microsomes, the proteins have no way of entering the lumen of microsomes once they are added. o No way to get across the membrane; must be co-translationally inserted into the lumen of the microsomes. · (b) Using microsomes in cell free protein synthesis assays, it can demonstrate the ribosomes can sit down on the surface of the microsomes and co-translationally insert the secretory protein into the lumen of the microsomes · In red is the signal sequence. · Once the protein is in the lumen of the microsomes the signal sequence is no longer require o Signal sequence is removed. Microsomes must be added before the first 70 AAs are synthesized and protrude form the ribosome o About 40 protrude and the remaining 30 are inside the ribosome This is so co-translational insertion can work

COPII coated vesicles (Fig. 14-8). What is Sar1 doing?

· COPII coat protein used to coat vesicles budding off rER and help escort the vesicles to the Golgi o by Anterograde vesicle transport. (outward) · COPII coat surrounds the vesicles budding off rER as it migrates toward the cis-Golgi o consists of several proteins Sec23/Sec24 and Sec13/31 complexes, and Sec16. o COPII binds to Sar1, a guanine nucleotide switch protein. § Which mediates the assembly and dis-assembly of the COPII coat. · Sar1 protein is the important regulatory protein needed for the formation of the COPII coat · Sar1 protein is soluble in the cytoplasm · A transmembrane protein in the membrane of rER, Sec12 is the exchange factor for Sar1 o The soluble form of Sar1 has GDP · After interacting with Sec12 in the ER membrane, Sec12 helps switch out the GDP for a GTP · Once GTP is bound Sar1 has a hydrophobic portion of its peptide sequences sticks out and anchors it to the membrane. · When Sar1 with GTP is bound to ER membrane, Sec23/Sec24(COPII coat) will assemble on to the Sar 1 proteins o As the vesicle is forming at the level of the rER o Sar1 tethers to the cytosolic leaflet of that membrane and then COPII will bind and form the coat

How are Histone Acetyl Transferases (HATS) directed to gene promoters? (Fig. 9-37b). GCN4 and GCN5 (Fig. 7-36b). Which one has the DNA binding and Activation domains? Which one is the HAT?

· Gcn4 is a leucine zipper type transcription factor and binds to the upstream activation sequence - a promoter proximal sequence usually found in yeast. o Uses DNA binding domain to specifically bind the UAS · Gcn4 with its activation domain also interacts/recruits co-activators - other proteins involved with initiation of DNA transcription for this particular gene · From yeast genetics, Gcn5 (a co-activators) was a transcription factor and important for activation of certain genes o Didn't know what Gcn 5 biochemical activity was with respect to the activation o Dave Allis - can we biochemically identify histone acetyl transferases § Enzymes that acetylate the lysine residues converting 30nm fibers into 11nm fibers § He was working in Tetrahymena, but the protein turned out to be an ortholog of GCN5. § First to identify using a very slick SDS-PAGE based assay · Diagram: Gcn4 uses DNA biding domain (Dark blue) to bind the UAS and it uses the activation domain to interact with several other co-activator proteins o Gcn5 is one of them § It is a DNA histone acetyltransferase. § Acetylates the nucleosome tails to open up the chromatin fiber of the promoter element. Other activators with histone acetyltransferase (HAT) activity: · CBP (CREB binding protein) a co-activator of CREB o CREB -a transcription factor § cAMP response element binding protein § element = DNA promoter segment in front of a gene that will respond to CBP and CREB o CBP - is a large protein about 400 kDa with multiple domains § One domain binds CREB § Other domains bind other transcription factors § One domain is a histone acetyltransferase · A HAT functionally related to Gcn5 · Largest subunit of TFIID o Called TAFII250 in higher eukaryotes o Also, a HAT activity · Many other nuclear HAT's

Pol I transcription (Fig. 9-51). What is a Nucleolar Organizer? What is a NoRC?

· Pol I - look at the promoter that drives transcription of the pre-ribosomal RNA o Occurs only in nucleolus o Promoter has an upstream control element at about -100 and a core element of +1 § Few base pairs down form +1 and several upstream of +1 · Recall that RNA Pol I's only job is to transcribe pre-ribosomal RNA inside the nucleolus. RNA Pol III can transcribe tRNAs and the small 5S ribosomal RNA, that then assembles into the large ribosomal subunit (LSU). · Pol I promoters have an Upstream Control Element at about -100, and a Core element which includes the transcription start site at +1. RNA Pol I transcription (in yeast): o Pre-ribosomal RNA transcribed in the nucleolus. o Core element (-40 to +5) includes the transcription start site. o Upstream control element (UCE) positioned at -155 to -60 · Protein factors necessary for rDNA transcription (in humans) o Upstream Binding Factor (UBF) o Selectivity Factor 1 (SL1) o TBP is a component of SL1 § same TBP as for Pol II o TIF-1A (RRN3p in yeast): § regulates the loading of Pol I o RNA Pol I itself (recall: many subunits) o nuclear actin and myosin - usually in muscle o deacetylase, SIRT7 (not for the histones) § involved with RNA Pol I transcription o Topoisomerase I § Relieves the excess supercoils o FYI: Cancer cells have a huge demand for ribosomes · Drug target for oncology: o RNA Pol 1 itself (many subunits) o Cancer cells have huge demands for ribosomes. · RNA Pol I does not use the General Transcription factors, but it does use the TATA binding protein (TBP) which is also a part of the SL1 protein complex. · Recall that like RNA Pol II, Pol I has several proteins subunits. · Molecular oncologists are designing drugs to selectively block Pol I, as cancer cells have a huge metabolic demand for the production of ribosomes. · The ribosomal genes are tandemly repeated: · In higher eukaryotes, these genes occupy the "Nucleolar Organizer" (NO) (term coined by B. McClintock) which is a secondary constriction on certain (mitotic) chromosomes. · In humans, NO's occur on chromosomes 13, 14, 15, 21, and 22. · (The primary constriction is the centromere.) o But those 5 chromosomes contain the secondary constrictions, the NO's. · Only ~50% of ribosomal genes are transcriptionally active. Why? o The other are facultative heterochromatin · Why are only half the rDNA genes expressed? Answer is unknown. o Inactive copies are condensed into facultative heterochromatin. · In mammals, for these silent rDNA genes: o Nucleolar Remodeling Complex (NoRC) blocks pre-initiation complex formation on the core promoter (keeps the genes silent) § NoRC recruits a histone methyltransferase · Histone H3 K9 is di- and trimethylated · this recruits Heterochromatin Protein 1 (HP1) o converting these particular ribosomal genes into the facultative heterochromatin o pRNA is a non-coding RNA. § also, a 250 nt RNA (called pRNA) is transcribed ~2000 bp upstream of the rDNA genes § pRNA helps target NoRC to the rDNA promoter · pRNA forms a RNA:DNA triple helix (double stranded) § NoRC with pRNA recruits DNMT3b that methylates CpGs in the upstream control element, thus blocking UBF from binding. · To recruit RNA Pol I. o partially explains how these 50% are silent. But WHY?

Pol III Transcription (Fig. 9-52).

· Pol III - the promoters are internal to the coding sequences for the particular genes. o Transcription factors bind to the promoters to recruit RNA Pol III to begin transcription at +1 the bent arrow Pol III promoters, on the other hand, are internal to the coding sequence (boxes A, B, and C)· RNA Pol III Transcription: · Promoters lie entirely within the coding regions of the tRNA and 5S rRNA genes. tRNA genes: · A box and B box for tRNA genes. · Once transcribed, the A and B box sequences are represented in the tRNA o necessary for protein translation. · TFIIIC binds A and B boxes (DNA) first. · TFIIIC then binds TFIIIB; TFIIIB then redirects TFIIIC to bind an extra 30 bp upstream of transcription start site (see next slide) 5S rRNA genes: · Recall, 5S rRNA genes are also repeated in tandem in metazoans · Transcribed OUTSIDE the nucleolus in metazoans, but INSIDE the nucleolus in yeast. · Metazoan 5S rRNA needs to move into the nucleolus for ribosome subunit (LSU) assembly. · Again, recall that Pol III has several protein subunits. tRNA gene: · TFIIIC binds to yellow segments (A and B boxes) · TFIIIB joins the complex. · Together TFIIIC and TFIIIB recruits RNA Pol III · RNA Pol III is about as big as the gene itself. · TBP is a subunit of TFIIB For the 5srRNA gene: · Small but it has an additional transcription factor TFIIIA · It binds to the C box bringing in TFIIIC and TFIIIB · TFIIIC redirected slightly to bring in RNA Pol III · TFIIIB also contains the TATA binding protein · TFIIIA is the first transcription factor characterized in detail · TFIIA has 9C2H2 zinc fingers o C2H2 are monomers 5S rRNA genes (continued): · TFIIIA = 1st transcription factor purified o Done by Don Brown o Won 2012 lasker award · TFIIIA first binds the C box within the 5S rRNA genes. · Then TFIIIC binds to TFIIIA, · and again TFIIIC is re-positioned back to -30 · TFIIIB then binds (contains TBP) brings in RNA Pol III · Interestingly, TBP is common to Pol I, II, and III. · Pol III can transcribe some TATA-box genes: · Ex. U6 gene o Encodes U6 snRNA o A TFIIIB like complex that binds to the TAT box; a further factor binds to the PSE further upstream and recruits RNA Pol III to transcribe the U6 gene · Does the U6 snRNA have a 7m-G cap? o Only transcripts transcribed by RNA pol II have a 7-methyl cap, so no it doesn't. Archaea are more closely related to eukaryotes than · prokaryotes in gene control. · Archaea have operons and produce polycistronic mRNAs (like prokaryotes). · But Archaea genes have TATA-like elements (like eukaryotes) o Prokaryotes do not usually have TATA like elements. · Archaea have a single RNA polymerase (like prokaryotes). · But the polymerase has ~13protein - subunits (like eukaryotes). · Some transcription factors are quite similar in AA sequence to those in eukaryotes and archaea. o e.g.: TBP and TFIIB

When things go bad: What forms of RNA surveillance occur in the cytoplasm? (Fig. 10-35a, b and c). With Xrn1 involved, where do you think mRNA decay occurs?

· RNA Surveillance in the Cytoplasm a) Nonsense mediated decay b) Non-stop decay c) No-go decay recruits XRN1 that degrades from 5' to 3'exonuclease activity Decay is likely in P bodies.

Vesicle fusion: SNARES, SNAPS, and Rabs. (Figs. 14-10 and 14-11).

· Sar1 protein is the important regulatory protein needed for the formation of the COPII coat · Sar1 protein is soluble in the cytoplasm · A transmembrane protein in the membrane of rER, Sec12 is the exchange factor for Sar1 o The soluble form of Sar1 has GDP · After interacting with Sec12 in the ER membrane, Sec12 helps switch out the GDP for a GTP · Once GTP is bound Sar1 has a hydrophobic portion of its peptide sequences sticks out and anchors it to the membrane. · When Sar1 with GTP is bound to ER membrane, Sec23/Sec24(COPII coat) will assemble on to the Sar 1 proteins o As the vesicle is forming at the level of the rER o Sar1 tethers to the cytosolic leaflet of that membrane and then COPII will bind and form the coat Transmembrane Cargo proteins are recruited to the forming vesicle · Interaction of Sar1 with GTp and Sec23/Sec24(COPII coat) · Notice the Sec24 protein is interacting with cargos within the ERs and the cargos have sorting signals that permit the proteins to interact with Sec24 and advance on to the Golgi o Only certain cargos are destined to move on to the Golgi, by way of interactions with the COPII coat o Vesicle is complete with addition of Sec 13/Sec 31 complexes (not shown). o Specific "sorting signals" in cargo proteins permit only certain cargos to advance to Golgi Once the vesicle blebs from the ER, the coat disassembles with Sec23 promoting the hydrolysis of GTP on Sar1 to GDT. · The COPII coated vesicle has detached from the ER o On the way to fuse with the cis-Golgi network membrane · Sec23 protein is working as a Guanine Nucleotide GTPase activating protein o Helps Sar1 hydrolyze the 3rd phosphate on GTP · Sec23 is acting as a GAP protein · Once Sar1 hydrolyzes the 3rd phosphate on GTP the entire coat assembly comes off the vesicle · Sec23 acting as the GAP has helped Sar1 hydrolyze the 3rd phosphate so Sar1 has GDP bound · With GDP bound the entire coat comes off the vesicle making it ready to fuse with the cis-Golgi network · Left hand side described the anterograde of the COPII coated vesicles from the rough ER to the Cis-Golgi membrane · Sec23 a protein in COPII coat, acts as a GAP to speed up the hydrolysis of GTP to GDP on Sar1 · With GDP bound to Sar1 the coat falls off the vesicle allowing it to dock to the cis- Golgi membrane. · The initial docking of the uncoated vesicle is by way of a Rab-GTP vesicle protein (a guanine nucleotide switch protein) o A protein in the vesicle membrane · Rab-GTP binds to a Rab-effector protein in the cis-Golgi membrane (first interaction between vesicle and Golgi) · SNARE proteins form coiled coils to hold the vesicle to the Golgi membrane - for membrane fusion o Many different SNARE proteins · Transport vesicle that just lost its COPII coat o Just ready to fuse with cis-Golgi membrane · Rab-GTP interacts initially with the Rab-effector protein in the cis-Golgi membrane o The initial docking · When the proteins come close enough together, the SNARE proteins form coiled coils and get the 2 proteins in contact together. · v-SNAREs - vesicle snares found in golgi membrane o form elaborate coiled coil structures I the yellow box · t-snares form an alpha helical bundle · "molecular Velcro" · When the transport vesicle is fused with the cis-Golgi o The cargo is released into the cis-Golgi lumen · The coiled coils in the SNARE proteins asre unraveled by complexes of proteins called NSF and alpha-SNAP so the coils can be reutilized