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Post by Vincent Bedami on April 27 at 10:04:46 AM

I have a review question that I have been struggling with and I'm hoping someone can help me understand.

You have discovered a new species of bacterium in Georgia, Baccillus Peachillis. You are studying the regulation of transcription in Bacillus Peachillis. The regulation of transcription in Bacillus Peachillis is exactly like that of E. coli, but the organism only expresses a single RNA polymerase. The RNA polymerase in Bacillus Peachillis is the only DNA-dependant RNA polymerase produced in this strain of bacteria.

You have generated the following data regarding data about RNA polymerase and ?68 from Bacillus Peachillis:
    -Like E. coli, RNA polymerase from B. Peachillisexists in a core and holoenzyme form.
    -The binding of ?68 to the core enzyme yields the holoenzyme.
         Holoenzyme?Core + ?68, Kd=0.26nM
    -RNA polymerase and ?68 are expressed constitutively in B. Peachillis at the following levels:
         Total RNA polymerase (core + holoenzyme) = 700molecules/cell
         Total ?68 = 700 molecules/cell
If the ratio of [holoenzyme]/[core]=4, what is the cellular concentration (in nM) of [holoenzyme], [core], and [?68]free?

Is the calculated values of [holoenzyme], [core], and [?68]free consistent with the cellular volume of a B. Peachillis cell of 1.0?m3? Why?

Transcription

    Long, 9 examples, 5 practice questions

  • Transcription is the synthesis of RNA using DNA as a template.
  • Transcription is a three-step process: initiation, elongation, termination.
  • Eukaryotic RNA polymerase I synthesizes rRNA, RNA polymerase II synthesizes mRNA, and RNA polymerase III synthesizes tRNA, 5S rRNA, and ncRNA.
  • Prokaryotes have a single multi-subunit RNA polymerase.
  • Precursor RNA molecules are converted into mature RNAs via several unique processing events.

Transcription

The process of making DNA form RNA is called:
  • Replication
  • Transcription
  • Translation
  • Reverse transcription
Which RNA polymerase is responsible for synthesizing mRNA?
  • RNA Pol I
  • RNA Pol II
  • RNA Pol III
  • RNA Pol IV
Where does RNA Polymerase II bind to initiate RNA synthesis?
  • BRE
  • TATA box
  • GC box
  • CAAT box
Where does the prokaryotic core RNA Polymerase bind to initiate RNA synthesis?
  • Pribnow box
  • TATA box
  • -35bp sequence
  • CAAT box
The process of removing introns and rejoining exons is called:
  • Capping
  • Splicing
  • Methylation
  • Polyadenylation

*These practice questions are only helpful when you work on them offline on a piece of paper and then use the solution steps function to check your answer.

Answer

Transcription

Lecture Slides are screen-captured images of important points in the lecture. Students can download and print out these lecture slide images to do practice problems as well as take notes while watching the lecture.

  1. Intro
    • Lesson Overview
      • Eukaryotic Transcription
      • Example 1
        • Eukaryotic Transcription: Initiation
        • Example 2
          • Eukaryotic Transcription
          • Eukaryotic Transcription: Termination
          • Example 3
            • Eukaryotic Transcription
            • Prokaryotic Transcription
            • Prokaryotic Transcription - Initiation
            • Prokaryotic Transcription - Elongation
            • Termination - Prokaryotes
              • Example 4
                • Example 5
                  • Post-Transcriptional Modifications
                  • RNA Processing
                  • RNA Processing - Capping
                  • RNA Processing - Splicing
                  • Example 6
                    • Alternative Splicing
                    • Example 7
                      • Example 8
                        • RNA Editing
                        • Example 9
                          • Intro 0:00
                          • Lesson Overview 0:07
                          • Eukaryotic Transcription 0:27
                            • Process of Making RNA from DNA
                            • First Step of Gene Expression
                            • Three Step Process
                            • Illustration of Transcription Bubble
                            • Transcription Starting Site is +1
                            • Transcription Unit Extends From the Promoter to the Termination Region
                          • Example 1 6:03
                          • Eukaryotic Transcription: Initiation 14:27
                            • RNA Polymerase II Binds to TATA Box to Initiate RNA Synthesis
                            • TATA Binding Protein Binds the TATA Box
                            • TBP Associated Factors Bind
                            • General Transcription Factors
                            • Initiation Complex
                          • Example 2 15:44
                          • Eukaryotic Transcription 17:59
                            • Elongation
                            • FACT (Protein Dimer)
                          • Eukaryotic Transcription: Termination 19:36
                            • Polyadenylation is Linked to Termination
                            • Poly-A Signals Near the End of the pre-mRNA Recruit to Bind and Cleave mRNA
                            • Mature mRNA
                            • Dissociate from Template DNA Strand
                          • Example 3 21:53
                          • Eukaryotic Transcription 25:49
                            • RNA Polymerase I Transcribes a Single Gene That Encodes a Long rRNA Precursor
                            • RNA Polymerase III Synthesizes tRNA, 5S rRNA, and Other Small ncRNA
                          • Prokaryotic Transcription 32:04
                            • Only One Multi-Subunit RNA Polymerase
                            • Transcription and Translation Occurs Simultaneously
                          • Prokaryotic Transcription - Initiation 38:18
                            • Initial Binding Site
                            • Pribnox Box
                          • Prokaryotic Transcription - Elongation 39:15
                            • Unwind Helix and Expand Replication Bubble
                            • Synthesizes DNA
                            • Sigma 70 Subunit is Released
                            • Elongation Continues Until a Termination Sequence is Reached
                          • Termination - Prokaryotes 40:17
                          • Example 4 40:30
                          • Example 5 43:58
                          • Post-Transcriptional Modifications 47:15
                            • Can Post Transcribe your rRNA, tRNA, mRNA
                            • One Thing In Common
                          • RNA Processing 47:51
                            • Ribosomal RNA
                            • Transfer RNA
                            • Messenger RNA
                          • RNA Processing - Capping 52:09
                            • When Does Capping Occur
                            • First RNA Processing Event
                          • RNA Processing - Splicing 53:00
                            • Process of Removing Introns and Rejoining Exons
                            • Form Small Nuclear Ribonucleoproteins
                          • Example 6 57:48
                          • Alternative Splicing 1:00:06
                            • Regulatory Gene Expression Process
                            • Example
                          • Example 7 1:02:53
                          • Example 8 1:09:36
                          • RNA Editing 1:11:06
                            • Guide RNAs
                            • Deamination
                          • Example 9 1:13:50

                          Transcription: Transcription

                          Hello and welcome back to www.educator.com.0000

                          Today, we are going to talk about the topic of transcription.0003

                          As an overview, we are going to talk about eukaryotic transcription,0009

                          then we will also talk about how that differs from prokaryotic transcription.0013

                          And then, we will focus on some of the post-transcriptional modification that are seen, 0017

                          the RNA processing, splicing, and editing.0023

                          What is eukaryotic transcription or what is transcription in general?0029

                          That is the making of RNA from a DNA sequence.0033

                          For eukaryotes, we are going to talk first.0039

                          We have RNA polymerase synthesizing RNA using one strand of DNA as a template.0042

                          This is the first step of gene expression and transcription is responsible for making rRNA, using RNA polymerase 1.0049

                          mRNA using RNA polymerase 2.0060

                          tRNA using RNA polymerase 3.0063

                          This is a 3-step process and it involves initiation, elongation, and termination.0067

                          We will talk about each one of these in depth.0073

                          First, we will show you what a transcription bubble would look like.0075

                          Here is our DNA, let us write in our RNA first.0106

                          In this case, I’m saying mRNA.0131

                          What we have here is the mRNA.0183

                          We will be making the proper hydrogen bonding.0189

                          We have DNA, we have it opened up.0196

                          This is what is called a replication bubble and it can vary in length.0198

                          What we have, we have our DNA opened up and we have our mRNA being synthesized off of a strand of the DNA,0206

                          that is acting as the template strand.0214

                          This right here would be called our template strand.0218

                          It has antisense.0230

                          This is what is called our coding strand and this has sense.0234

                          Sense is also known as positive, antisense is known as negative orientation.0245

                          Our mRNA will be sense as well because it is going to be complementary to the template so it will have to be the opposite.0250

                          Sense or negative.0262

                          By the way, this is all occurring inside of RNA polymerase, big RNA polymerase going through.0267

                          Since this is mRNA, this is RNA pol-2.0277

                          This mRNA has a sense or negative strand, which is why we can talk a little later on in these units, 0285

                          why we can use antisense RNA or siRNA, silent interfering or small interfering RNA,0295

                          which can bind to and degrade mRNA which can be a way that we can regulate gene expression.0302

                          Let us continue on.0314

                          Let us talk about eukaryotic transcription.0317

                          We have the transcription start site which we call the TSS, is being found at the +1 position.0319

                          The promoter and most of our regulatory regions are upstream or to the negative side.0329

                          Our transcription and termination regions are downstream or to the positive side.0334

                          Our transcription unit, the entire unit, extends from the promoter to the termination region.0340

                          It will include such things as a GC box, a CAT box, TATA box, BRE initiator, DPE.0352

                          I will tell you what each one of these in the following slides.0358

                          Let us draw a typical eukaryotic transcription unit. 0364

                          Let us start with our initiator or our +1 site.0372

                          These dots mean that there is extra DNA in between.0382

                          This is roughly what we are going to see.0432

                          This would be, what do we have here?0437

                          Let us see, the initiator, that is where we are going to start transcription.0467

                          What I can do is I can draw this as if this would be the RNA.0473

                          This is the pre-mRNA.0478

                          It is going to start here and it is going to go all the way down.0484

                          It is going to go past this stop sequence, all the way down further.0490

                          This would be a UAA, UAG, or UGA.0496

                          This stop codon, that is where the translation stops but not where transcription stop.0504

                          We usually transcribe further pass the stop codon.0510

                          But this is as far as I’m going to show you.0514

                          I'm going to start right here, this would be your AUG.0516

                          If we think about the protein being produced, protein is going to go like this.0521

                          From N terminal to C terminal, it is going to be that long, between the start and the stop codon.0531

                          This would be translation between here and here.0535

                          Translation between here and here, transcription.0545

                          What do we have first?0553

                          We have the initiator, that is the +1.0554

                          Upstream of the initiator, at about -25 bases, is the TATA box.0556

                          The TATA box has a conserve sequence of TATA, AAA in humans.0563

                          This is the site of our TF2D binding as well as the TATA binding protein.0576

                          The BRE is where we have TF2B binding.0588

                          BRE is the B recognition element.0598

                          This is found directly upstream of the TATA box.0604

                          At about, maybe 80 base pairs upstream, we have what is called the CAT box.0608

                          This is where we have random transcription factor binding.0614

                          Upstream of that, at about -100 base pairs, this is where we find, by the way the CAT box has around that CAT sequence.0622

                          The GC box is roughly GGG, CGG sequent.0634

                          This is where we find transcription factor binding, but specifically it is protein SP1.0647

                          This DPE is the downstream promoter element, that we find at about +25 bases.0657

                          Remember, this in the black is DNA, in the red is mRNA, and then we would have the protein down here.0681

                          Only about, let us say, 10% of human promoters actually have a TATA box which can also be called the hogness box.0698

                          Most promoters do not have this CAT box.0710

                          But the ones that do, it is very important for the frequency at which you will go through transcription.0714

                          The GC box is all required for transcription and are bound by the transcription factor SP1.0723

                          Typically, genes have either a TATA box or a DPE, downstream promoter element, but you would not have both.0731

                          From the BRE to the DPE, this is called the core promoter.0744

                          The GC and CAT box are part of the proximal regulatory element, sometimes called the proximal control element.0770

                          And then, very far upstream, we can find enhancers.0796

                          Enhancers are part of the distal regulatory element or the distal control element.0805

                          How this might work is, let us say, if this is DNA, we can have enhancer sequences way upstream in here.0822

                          Remember, here is our core promoter, let us say, our +1 site around here.0835

                          This could be thousands upon thousands of bases away, this enhancer,0842

                          but proteins can end up bringing these too much closer together by bending the DNA,0846

                          so that you can bring these sequences close together, so that you can enhance the frequency of transcription.0856

                          Let us go on with initiation.0868

                          How does transcription start?0870

                          We have RNA pol-2 binding to the TATA box, to initiate RNA synthesis.0874

                          The TATA box is located, it is centered around that -25 base pair mark.0882

                          How do we get this to happen?0890

                          First off, we have TATA binding protein, TBP binding the TATA box.0894

                          Then, we have what are called TAF, TATA binding protein associated factors, binding to TBP.0901

                          When we have TBP and TAF bound, we call that transcription factor 2D or TF2D.0914

                          Then, we get many more TF2 binding, ABFENH.0924

                          The TBT, TF2, and RNA pol-2 together, form what is called the initiation complex.0931

                          It will be bound up upstream of the initiation site so that everything can start.0938

                          Let us look and see what that looks like.0944

                          Here is our DNA, here is our TATA box at about -25 base pairs.0960

                          Let us say this is our initiation spot or our +1.0977

                          What we have is TATA binding protein binding here.0986

                          TF2D binding that.0998

                          We have RNA pol-2 coming in.1005

                          And then, we have a bunch of our TF2, the rest of our TF2 eventually binding, TF2, ABEHS and F.1017

                          Once again, our core promoter runs through that DP element, if we have it.1039

                          It is a core promoter.1053

                          And then, our gene would be starting, once we hit the actual the start codon which would be shown up in the RNA.1059

                          Maybe the gene runs over here.1073

                          After we have the initiation, we have all the binding of the factors, we can then go through elongation.1082

                          This is when we can actually leave the promoter region.1087

                          RNA polymerase 2 can leave the promoter region and start synthesizing in a 5 prime to 3 fashion,1091

                          making us that nice pre mRNA transcript.1099

                          Very important, we have the function of a protein called FACT, it is a dimer meaning there is 2 subunits.1103

                          This allows RNA polymerase to transcribe through the regions associated with histones and with secondary structure.1113

                          This is really important that transcription needs to occur all the way through a gene, 1120

                          no matter if that gene happen the bound by a histone or let us say the DNA has some sort of secondary sequence or secondary structure.1125

                          It is really hard to get through all this.1141

                          But FACT can basically come in here and roll this out nice and straight.1146

                          As RNA polymerase is bound and moved in this direction, it is coming to no places that are going to stop it.1156

                          The final part of eukaryotic transcription which is termination,1181

                          is brought about by an RNA processing event called polyadenylation, adding a poly-A tail to an mRNA. 1190

                          Poly-A signals which found in the DNA as the AAU, AAA, which is found near the end of the pre mRNA transcript,1199

                          will recruit CPSF and CSTF protein complexes.1210

                          These will bind to the mRNA and cleave the mRNA, releasing it from RNA polymerase 2.1216

                          Once that has been cleaved, poly-A polymerase, sometimes short form is PAP.1226

                          Then, we will add about 200 A, adenosine nucleotides, to the newly cleaved 3 prime end, 1236

                          ultimately, finally making it a mature mRNA.1244

                          At this point, it is already gone through what we are going to see later as the capping and the splicing of the mRNA.1248

                          The final processing event is the polyadenylation.1259

                          At that point, the mRNA is mature and can leave the nucleus and go through translation in the cytoplasm.1262

                          Once the mRNA has been released, we have XRN2 endonuclease digesting any of the excess hangover or overhang, 1273

                          that is still connected to RNA pol-2 which is connected to the DNA still.1286

                          XRN2 continues to digest until it reaches RNA pol-2 and causes RNA pol-2 to release or dissociate from the DNA.1292

                          As I said, the matured mRNA can leave to the nuclear pore and go to the cytoplasm to be translated.1303

                          What is termination look like, let us see.1314

                          Here is our 5 prime, we are going to say this is our mRNA.1318

                          We already have the cap.1328

                          Maybe this is our start codon.1338

                          Maybe this is our stop codon.1343

                          After the stop codon, we will find that poly-A signal which is the AAU, AAA.1348

                          Then, we have a gap of about 10 to 30 bases before we get to a part that is G rich, guanine rich, or G and U rich.1363

                          Let us just keep going, then we have some excess.1393

                          This is DNA.1419

                          This must be an RNA pol-2 in here transcribing.1436

                          It is going in this direction, 5 prime to 3 prime.1442

                          What we have is we have the CPSF protein complex, binding at that poly-A signal.1450

                          We have the CSTF complex binding at the G rich or GU rich portion.1461

                          What we have is we have our cut happening here, that is going to release the mRNA.1472

                          And then, we can add the poly-A tail to the end.1485

                          Now, if we just think that that has been cut, what we then have is XRN2 coming in.1490

                          What it is going to do is, it is going to start chewing this up.1501

                          It is an exonuclease, chew it up, until it gets all the way up to RNA pol-2.1504

                          And then, that is going RNA pol-2 to dissociate from the DNA, that will come off.1513

                          In which case, we have terminated transcription.1519

                          That is how transcription is terminated for RNA pol-2, for transcription of mRNA in eukaryotes.1522

                          And then, what we have done then, we will have all of our A and that is from the poly-A polymerase.1531

                          If we want to talk about the other two eukaryotic RNA polymerases involved in transcription, let us do that.1554

                          RNA pol-2 is what we have talked about so far, that is what synthesizes mRNA.1563

                          Remember, mRNA is the type of RNA that will go on to make proteins.1568

                          RNA pol-1 transcribes a single gene that will encode a long rRNA precursor, long ribosomal RNA precursor.1574

                          The way that they terminate transcription, they will go through the same type of initiation and elongation,1589

                          but this will terminate in a different way.1595

                          It does that by, it has an actual sequent that causes termination.1597

                          The transcription is terminated with response to specific termination sequent found in the DNA that is being transcribed.1605

                          That is how we terminate transcription.1616

                          Just because we really only look at the transcription unit of pol-2, I will just write out pol-1 is here.1619

                          If we see on the DNA, we have our +1 site, the initiation.1629

                          What we have is our core promoter, that runs from about +20 to -45 bases.1642

                          Then, we have a nice gap.1655

                          Then, at about -100 to -150, remember this is DNA, this is called the core promoter.1658

                          This is the UCE or upstream control element.1672

                          The upstream control element is bound by a protein called UBF.1677

                          That is just the upstream binding factor.1685

                          The core promoter is bound by a TBP, RNA pol-1, that is both going to be upstream of the initiation site.1687

                          Also, we have SO1 protein binding.1707

                          What we are going to do is, we will make a nice pre-rRNA.1713

                          Our rRNA is made in the nucleolus, specifically.1727

                          It is in the nucleus and it is in a specific region of DNA and protein called the nucleolus.1732

                          That is what the transcription unit of an rRNA gene looks like.1744

                          Let us talk about the termination of transcription for RNA polymerase 3.1754

                          Then, I will draw you the transcription unit again.1760

                          RNA polymerase 3 synthesizes mainly tRNA, as well as 5s rRNA and some other small ncRNA which is non-coding, 1764

                          it does not code for anything.1775

                          It is not going to be a protein.1777

                          The way that RNA polymerase 3 transcription is terminated, it is in response to certain sequences found in the newly synthesized RNA.1780

                          Pol-1 stops based on sequences found in the DNA.1794

                          Pol-3 stops based on sequences found in the RNA.1798

                          What does a transcription unit look like for a RNA pol-3?1802

                          Let us say this one is going to be making tRNA.1810

                          We have our DNA.1814

                          We have our +1 site.1821

                          Upstream of that, we have TBP binding.1826

                          TF3B binds that and RNA pol-3 will bind that.1835

                          Remember, this all has to happen upstream, on the negative side of the initiator, that way, transcription can start on time.1855

                          Another couple of things that are found, that is unique to the tRNA is that upstream, we have two different boxes.1867

                          We have box A and box B, found directly next to each other.1879

                          They are bound by a transcription factor called TF3C.1886

                          What this would look like, we have our tRNA being made, 5 prime.1900

                          This is our pre-tRNA.1907

                          That is what the transcription unit would look like for a tRNA gene.1914

                          That is how we go about eukaryotic transcription.1926

                          Let us talk about prokaryotic transcription.1932

                          Prokaryotic transcription is carried out by a single RNA polymerase.1936

                          No matter what type of gene it is transcribing.1943

                          As we saw in eukaryotes, there are three different types.1947

                          We have RNA pol-1, 2, and 3.1952

                          Each is responsible for synthesizing a different type of RNA.1954

                          We really have one in prokaryotes.1957

                          We have what is called our core enzyme.1961

                          That is a multi-subunit complex made up of an α subunit, a β subunit, a β prime subunit, and an ω subunit.1963

                          This is going to bind non-specifically the DNA and it will synthesize in a 5 prime to 3 prime fashion, as we are familiar with.1973

                          For it to be nice and fully active, we need this to be in a holoenzyme,1984

                          meaning the core enzyme + another factor called the sigma factor or the sigma70 factor.1992

                          Together, that can bind the proper promoter region on DNA that is what sigma70 is important for.2002

                          The sigma70 subunit allows the core enzyme to recognize the promoter regions on DNA.2010

                          We are allowed to then transcribe as normal.2018

                          Also, one thing that is very different about prokaryotic transcription vs. eukaryotic is that2022

                          transcription and translation will be occurring at the same time.2029

                          Remember, prokaryotes do not have a nucleus.2032

                          You do not have to wait for the RNA to be synthesized, sit out of the nucleus, and then translate it.2035

                          This can be happening all at the same time.2041

                          What does prokaryotic transcription look like then?2046

                          This is the DNA, we have RNA polymerase.2066

                          We have the RNA being made.2090

                          This transcription bubble, this is what we call that, that is about 14 base pairs.2101

                          It is about 14 base pairs that is in single stranded form.2113

                          What we have is, as RNA polymerase going in that direction, it is continuing to make RNA.2119

                          No RNA coming out.2127

                          We can also have ribosomes on here, multiple ribosomes making many different copies of the protein from the same RNA.2130

                          What we see here is that, all the ribosomes will be traveling in that direction.2148

                          This one will be putting out a protein.2157

                          This one, the longer protein.2164

                          These are all protein.2171

                          This will be the N terminal tails.2176

                          Those will be N terminal tails.2181

                          This can be happening at the same time in a prokaryote.2183

                          It will not happen at the same time in a eukaryote.2185

                          You have to have transcription and translation separate because transcription always happens in the nucleus,2189

                          translation happens in the cytoplasm.2195

                          The 16S rRNA and the 30S subunit of the ribosome which we are going to talk about next time during translation,2200

                          then will hydrogen bond with the sequence called the shine gargano sequence which is AGG, AGG, that is the consensus sequence.2217

                          Not always exactly that, but the closer it is to this, the better binding affinity it has.2234

                          The shine gargano is about 8 base pairs upstream of the start codon.2241

                          Let us say the start codon is right here.2252

                          Your new ribosomes will start binding just at that shine gargano in that sequence right there.2259

                          As they move pass that start codon, then it will start making a little bit of that protein at the time.2266

                          The 30S will bind and then actually the 50S will come and join.2274

                          We will talk more about this, when we talk next time.2285

                          And that allows us to have the full 70S ribosome, and allow us to start translation.2289

                          Initiation of prokaryotic transcription, it happens when we have the RNA polymerase holoenzyme binding the promoter,2301

                          the core enzyme + the sigma70 factor.2309

                          It binds at the -35 base pair sequence.2312

                          It is the TTG, ACA, that is roughly the conserve sequence.2317

                          The prokaryotic version of the eukaryotic TATA box is called the pribnox box and that is found about 10 base pairs upstream.2323

                          It has a very similar sequence, TAT AAT.2333

                          Our human TATA box is TATA AAA.2337

                          This is the site of initial DNA unwinding, melting the helix which creates the transcription bubble, 2341

                          allowing the synthesis to occur once we hit that initiation site or the initiator site.2347

                          For elongation, what needs to happen is that holoenzyme, the core enzyme needs to be able to leave the transcription start site.2356

                          RNA pol continues to unwind the helix locally, expanding the replication bubble.2365

                          Think of it as sucking in the DNA, instead of moving.2370

                          RNA polymerase is going to synthesize 5 prime to 3 prime.2375

                          After 10 base pairs are synthesized, this can take many times.2379

                          In fact, it usually does take several trials before they can even get to 10 full base pairs.2384

                          Once they do, the sigma70 subunit is released.2390

                          It releases from the full holoenzyme, not just the core enzyme.2397

                          The core enzyme can leave the promoter and continue on.2402

                          Instead of sucking the DNA to it, it can start traveling along the DNA.2405

                          Elongation will continue until a termination sequence is reached.2409

                          What about that termination sequent?2418

                          Termination can be, this is ρ.2420

                          This can be ρ-independent which is the most common, or ρ-dependent.2425

                          I’m going to tell you what ρ-independent termination is or what it looks like.2430

                          If we have our DNA, we have our RNA pol in here, going this way.2436

                          We have our RNA coming out.2473

                          For ρ-independent termination, we have to find what are called inverted repeats and they are GC rich.2491

                          We find them in the DNA.2501

                          We have an inverted repeat here and here.2503

                          When we have already gone to the transcription over here, we have these inverted repeats on the RNA.2506

                          These are going to be found after the stop codon.2515

                          Let us say the stop codon is right here, that is the stop codon.2519

                          Let us make this look nicer and make it longer.2532

                          We have these inverted repeats that can hydrogen bond to each other because they have the same sequence.2544

                          What that does, what we have happening is this comes together.2553

                          These are the inverted repeats, they are hydrogen binding together.2570

                          Once again, this is our 5 prime of our RNA.2575

                          These inverted repeats will hydrogen bond together, making it what looks like a tennis racket.2581

                          This tennis racket has a very GC rich because these are inverted repeats or GC rich.2587

                          This tennis racket formation will cause the RNA still with the RNA pol attached to it, to pull completely off of the DNA.2600

                          This is what we call ρ-independent termination.2613

                          ρ-dependent termination, it is dependent upon the ρ protein.2616

                          It basically cuts it off of the RNA polymerase.2621

                          ρ-independent is this way, it is the inverted repeat, tennis racket handle, pulling away.2627

                          We have talked about transcription and let us compare the nucleotide sequences of the coding strand,2641

                          the template strand, and the nascent strand, so we see what is going on.2645

                          Let us first start with DNA.2649

                          If it is ATGC, ATGC, AAA, this is DNA.2659

                          Our DNA is complementary, this is going to be a TACG, TACG, TTT.2678

                          This strand is called the coding strand.2695

                          Otherwise known as the non-template strand.2702

                          It has a polarity of synth or positive.2712

                          This strand is the template strand or the non-coding strand.2725

                          It has a polarity of anti-synth or negative.2741

                          Our RNA is made from the template strand.2753

                          It has to be complementary to the template strand.2761

                          What we have would be AUGC, AUGC, and then, AAA.2769

                          This is the same polarity as the coding strand.2787

                          This is the synth or positive.2793

                          As we can see, it has the exact same sequent as the coding strand except for the U's are found, instead of the T's.2799

                          Your RNA will have the exact same sequence except U’s switch for T's, as the coding strand.2817

                          It will be the exact complement to the DNA strand.2824

                          Once again, the U’s switch for the T's.2830

                          We talked about transcription.2838

                          Let us talk about what happens after transcription.2839

                          What happens to that RNA?2842

                          These are what are called post-transcriptional modifications.2844

                          You can post-transcriptional modify your rRNA, your tRNA, and your mRNA.2849

                          You are going to do that in each of 3 different ways.2855

                          All of these share one thing in common, that they are all made by processing the mature ones,2858

                          they are all made by processing and modifying long precursor molecules.2864

                          We have the first one, ribosomal RNA or rRNA.2873

                          It is made by processing precursor molecules called pre-rRNA.2879

                          In prokaryotes, the 5S, the 16S, and the 23S rRNA are all made from a single pre-rRNA molecule.2894

                          It should always say pre-rRNA on this slide.2911

                          In eukaryotes, the 5.8S, the 18S, and the 28S rRNA are all made from a single pre-rRNA molecule.2915

                          The 5S rRNA in eukaryotes is made from a different rRNA molecule.2930

                          In general, what we see, we have three different RNA being made from a single precursor molecule 2936

                          that eventually gets chopped up, to make your three separate rRNA.2942

                          For the tRNA, we once again make it by processing a longer precursor molecule.2949

                          What we have to do to the tRNA, once again is different than the rRNA or the mRNA.2956

                          What we need to do is we will remove sequences in both the 5 prime and 3 prime end.2962

                          Sometimes we have an intron present in the anti-codon loop.2970

                          If we do have that, we need to remove that.2975

                          Very importantly, we have a CCA sequence, cytosine-cytosine-adenosine, that is added to the 3 prime tail.2980

                          We will talk why that is so important, next time in translation.2992

                          Briefly, the CCA part is where the amino acid is attached to the tRNA, 2996

                          so that it can be brought into the ribosome and adds it when the proper codon, as seen from the mRNA.3006

                          We have a bunch of bases, specific bases that are modified through very specific places, positions in the tRNA.3018

                          Examples of that can be methylation of bases or urylation of bases.3030

                          It is all happening at specific spots on specific bases.3036

                          Finally for processing mRNA, they get made once again from a long precursor molecule.3042

                          This, once again, it is a pre-mRNA.3048

                          There are three main things that happens to an mRNA.3052

                          First, you cap it.3054

                          Second, you splice it.3056

                          Third, you add a poly-A tail.3059

                          First thing is first, you add the 7 methyguanosine triphosphate or 7 GMTP cap to the 5 prime end.3062

                          This is nuclease resistant, the mRNA cannot be eaten up by nucleases before it is turned into a protein.3071

                          You also have the addition of the poly-A tail.3082

                          You add about 200 adenosines to the 3 prime end via the enzyme polyadenylate polymerase or poly-A polymerase.3086

                          We have already talked about it being called PAP.3094

                          Also, we go through splicing.3098

                          We remove introns and rejoin the exons, to have a contiguous sequent.3100

                          It is important that we see that histone transcripts contained 0 introns.3106

                          Most eukaryotic genes do contain introns.3115

                          It could be from 1 to upwards of 50.3119

                          Most do contain introns.3123

                          Let us talk about each one of these processes or mRNA editing or processing.3131

                          Capping, it occurs as soon as that newly synthesized pre-mRNA starts to come out of RNA pol-2.3140

                          It is the first event and it involves the addition of this 7 GMTP cap, added to the 5 prime end of that mRNA.3150

                          This is really unusual that it is added in a 5 prime to 5 prime linkage.3159

                          This is highly nuclease resistant.3166

                          You cannot even cleave that cap off very well.3169

                          It is happening right here.3173

                          Here is the mRNA, here is the cap.3175

                          Splicing will be occurring next and that is removing introns, rejoining exons.3181

                          That is necessary for most eukaryotic mRNA to proceed all the way through to translation.3187

                          If you do not go through splicing, you likely will not get a fully functional protein.3192

                          Splicing is catalyzed by the spliceosome, we are going to talk about that in the next few slides.3200

                          What the spliceosome is, it is in association or an aggregation of small nuclear RNA and proteins.3205

                          They associate to form what are called small nuclear ribonucleoproteins or snRNP.3216

                          Let us talk about how splicing occurs.3227

                          First, we have our snRNP associating, that facilitates being able to splice out introns.3231

                          How does it start?3239

                          I’m going to tell you all this in words first and then we are going to go over the picture.3242

                          snRNP bind to the 5 prime splice donor site.3247

                          When I say donor site, that means where the splicing is going to occur.3252

                          It binds to the 5 prime splice donor site and the 3 prime splice acceptor site.3256

                          That means, if we have a long piece of RNA, we have exon 1, we have intron1, then, we have exon2.3263

                          We will have splice site, it is right here, each one of these lines.3278

                          The snRNP will bind right here and right here.3284

                          They will bring them together like this.3289

                          This is the intron, this is exon2, this is exon1.3295

                          snRNP are all bound right here.3304

                          It brings those close together.3309

                          The 2 prime hydroxyl of the branch site adenine of the intron, attack the 5 prime phosphate at the splice donor site.3314

                          What is important is that there is a very important adenine in the intron and 3324

                          that is what we call the branch point or the branch site.3331

                          The 2 prime hydroxyl of that adenine will attack the 5 prime at the splice donor site.3337

                          It will attack over here.3346

                          The A is here, it will help attack there.3348

                          This will form a specific phosphodiaster bond in a 5 prime to 2 prime confirmations that creates a structure known as a lariat.3352

                          What it is going to look like, I am drawing it here but we will see a nice picture is.3362

                          What we have is, what looks like this.3368

                          This being exon1, this is being exon2, this being the intron.3377

                          This is what we call the lariat.3385

                          The 3 prime hydroxyl of this exon1, this is a 3 prime hydroxyl, will attack the 5 prime phosphate.3394

                          Here is the phosphate at the splice acceptor site, which allows that lariat to basically be cut and released.3407

                          The lariat gets released and therefore the intron has been excised.3432

                          Now, all you have to do is pull the two exons together, re-ligate that nice gap 3437

                          between the 3 prime free hydroxyl and the 5 prime free phosphate.3443

                          Now, you have a nice contiguous sequence again.3448

                          If that is done to all the introns you want out of there, it is considered able to be mature.3452

                          After adding the poly-A tail, you can send it on its way out into the cytoplasm to be translated.3461

                          Here is an example with the pictures and with more specifics on what the snRNP are.3469

                          Here we go, our U1 SNIRP attaches at the 5 prime splice donor site.3476

                          It will attach right here.3492

                          This would be U1.3494

                          Our branch point binding protein will bind to the branch point adenine.3500

                          Here is that branch point adenine.3507

                          U2 will bind to that branch point and the branch point binding protein will leave.3513

                          The important thing to point out right here is that, for U1 to bind, it needs the SR protein.3524

                          SR proteins are very important because they can be, they are regulation, whether up or down,3530

                          they can be regulated often in cancer cells.3538

                          Now that U1 and U2 are bound, we have U4, U5, and U6 attaching as a complex and basically going like this, 3543

                          bringing the exons closer together and looping out that intron.3554

                          This is happening here.3559

                          U1 releases the new before it comes off.3562

                          We have the formation of the lariat which is down here.3567

                          We have the 3 prime hydroxyl of this free exon1 which will be over here, it will be free, 3575

                          attacks this 5 prime splice site over here, which would be there.3584

                          It cuts that lariat off.3590

                          It cuts that off and what we have is the lariat.3593

                          The snRNP can release, the introns is being removed.3597

                          The exons, all they need to do is have them be re-ligated together.3601

                          From there, we have the intron being released and the exons being ready to be ligated together.3608

                          What is important is that, I have only shown you two different exons separated by a single intron.3616

                          We can have this phenomenon called alternative splicing occur.3621

                          Alternative splicing is a regulatory gene expression process that can make many different types of mRNA 3627

                          which can lead to different types of protein from a single pre-mRNA transcript.3634

                          For example, here is your DNA.3642

                          In this gene, you have 5 different exons.3645

                          When you transcribe, this is transcription, you will get your RNA.3648

                          If everything goes well, you have all 5 exons.3658

                          All that genetic material in between, these are introns.3660

                          This is intron1, intron2, intron3, intron4.3670

                          You can alternatively splice this to make multiple different mRNA.3680

                          For this mRNA, all we did was splice out all 4 introns and smooch all 5 exons together.3685

                          In this one, we spliced out the 3 introns but we did not include exon5.3692

                          For this one, we included exons 1, 2, 3, and 5 but not 4.3700

                          You can think of there are many different ways to do this.3706

                          Once we go through translation, the mRNA that had all 5 exons will give you this protein, protein A.3709

                          The one that had these specific form, 1, 2, 4, and 5, will give you a different protein, protein B.3719

                          And then, this one that had exons 1, 2, 3, and 5 will give you yet another protein, protein C.3727

                          You can see you can make many different proteins from a single gene due to alternative splicing.3734

                          That is why the whole one gene-one protein hypothesis had to be thrown out the window,3740

                          that was given by Beadle and Tatum in 1941 because of such things seen through alternative splicing.3746

                          We can make, for example here, 3 different proteins from the same gene.3753

                          We can make this polypeptide, we can make many different polypeptides from the same gene.3760

                          Let us talk about alternative splicing, I will give you an example of it.3776

                          We have SV40 large T and small T antigens.3781

                          SV40 is a simian virus 40 and it can affect humans as well.3784

                          Let us draw this out.3792

                          We have pre-mRNA, we have exon1, intron, exon2.3795

                          Here we find a stop codon.3857

                          We have our 5 prime splice site, right there and right there.3873

                          We have our 3 prime splice site, right there and right there.3878

                          We have a weak 5 prime splice site right there.3890

                          With the normal stop codon being here.3911

                          When the mature mRNA is made from this top one, the mRNA from the top one, 3923

                          what we have is exon1, the intron splice out, and exon2.3947

                          What this is going to do is, from here we can do translation.3983

                          We can get a protein and it will be this length.3998

                          This is this one.4017

                          This is called the large T antigen, this protein.4028

                          If we talk about this one, what we see is we have exon1 perfectly fine.4045

                          Instead of splicing properly at that 5 prime splice site, it splices at this weak 5 prime splice site.4078

                          What we end up seeing is we have that exon, we also have a little bit of that, it goes to that R, INTR and then exon2.4094

                          Remember, we have a stop codon in this intron.4114

                          With this stop codon, we do not see in this first mRNA because we have spliced it out.4120

                          What we see here when the protein is made, we have our NH2 and it only goes to here.4127

                          This is our small T antigen.4144

                          It is a truncated protein that does not include any of exon2.4149

                          This is not necessarily a way of alternative splicing but this is a way that it is technically alternative splicing4154

                          because you have a different protein from the same gene.4163

                          Although, this was not necessarily how I showed before with multiple exons and you are rearranging that.4167

                          Another example of alternative splicing, more or so of what I talked about before is that we have this protein called CD44.4176

                          These proteins are found on the outer cell membrane in mammalian cells.4184

                          They have 20 different exons with more than 30 different splice variant which can lead to about 20 different functional proteins.4189

                          We have specific isoforms of CD44 being found primarily on some cancer cells.4201

                          We can see that some type of alternate splicing can affect, if you may get a disease or not.4208

                          Actually, what we see is that about 10 to 15% of human disease is actually caused by defects in alternative splicing events.4216

                          More than 65% of all eukaryotic genes undergo alternative splicing.4228

                          We have of 65% of all the genes that we are making, they have a chance to have an alternative splicing defect and that can lead to disease.4234

                          As I mentioned before, those SR proteins, those are essential for splicing.4244

                          They are found in higher quantities in cancer cells than in normal cells.4251

                          There is more alternative splicing that can be done in cancer cells to,4255

                          maybe get them a certain splice variants such as these that they really need.4258

                          Just the last couple things I want to tell you, in terms of RNA processing, let us look at RNA editing.4268

                          There are 3 different ways that you can edit in RNA.4277

                          The first one, I’m going to give you an example of this on the next slide, is by using guide RNA or gRNAs.4280

                          These guides RNA insert two uracil nucleotides into the mRNA.4287

                          The whole reason it does that is to do a frame shift, it shifts the reading frame.4292

                          What is required for gRNA, we need an endonuclease, a 3 prime tedase or terminal uridyl transferase.4298

                          We need UTP and we need an RNA ligase.4309

                          The other way of editing RNA that we are going to talk about, can happen n two different ways.4313

                          Deamination is the way of editing it.4319

                          The first way of looking at it is the more common way which is site specific deamination, that is where we will deaminate.4323

                          We would take off that NH2, from the cytosine in a CAA stretch to make it a UAA.4332

                          That UAA is now a stop codon.4340

                          The stop codon will truncate the protein.4344

                          This deamination occurs via a cytidine deaminase.4349

                          We have proteins in our body called aid, a type of cytidine deaminase, that can truncate proteins.4356

                          This is our own body’s way of fighting against the HIV virus.4370

                          It specifically protects us against the HIV virus trying to make their new transcripts.4375

                          A rarer type of deamination is enzymatic deamination.4388

                          An example of that is turning an adenosine into inosine via adenosine deaminase.4393

                          Instead of A pairing with T, I would pair with C.4401

                          Or instead of A pairing with U, I would pair with C that will affect the proper tRNA that will bind to the codon in the mRNA.4406

                          You will get possible a different amino acid input into the growing polypeptide, 4418

                          instead of the proper one without this deamination occurring.4425

                          Let me give you this example of the guide RNA.4431

                          If this is what the mRNA looks like.4434

                          The sequence is GAG, UAUA, CCU.4442

                          The guide RNA might look something like this.4458

                          This right here, these a’s, are looped out.4504

                          Those are looped out, right there.4523

                          What it is going to do is it is going to try to add in these A’s to make it relevant on the mRNA.4524

                          What we have is, those A’s being looped out.4533

                          We have an endonuclease coming in here.4537

                          What we have is if this is the mRNA again, we have the GA blank blank, GUAUACCU.4547

                          Then, we have the gRNA.4565

                          We have the endonuclease coming in and cutting this backbone.4585

                          If we look at the backbone, it looks like this now.4589

                          That is going to be empty.4601

                          The next step would be, we have tedase coming in and adding our two U’s and RNA ligase sealing the nick.4608

                          What we end up seeing is.4630

                          What happens here is that we have lengthen this.4671

                          Now, we have possibly affected the reading frame.4674

                          We have 3 possible reading frames, if we look at mRNA.4680

                          We can say this, let us write our mRNA down here.4684

                          We will talk more about reading frames, next time in translation.4692

                          If we say that here is our original.4696

                          If this was our original reading frame, we call the ORF1.4706

                          We have another reading frame that could be ORF2.4712

                          We have a 3rd reading frame that could be ORF3.4718

                          This can affect when you will see a particular start-stop, and any other codon in between.4722

                          If you move the reading frame in other than bases of multiples of 3, 4733

                          you will affect the reading frame going from ORF1 to ORF2, or ORF1 to ORF3.4739

                          This can affect the total length and composition of your protein.4744

                          Guide RNAs can definitely edit mRNAs which will edit the final protein product.4749

                          That is the end of today’s lecture on transcription.4757

                          I hope you join me next time for our unit on translation.4760

                          Thank you once again for joining us on www.educator.com and I hope to see you again soon.4764