Molecular Biology 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 molecule2936

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 and3324

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 gap3437

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 mRNA3627

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