Molecular Biology Translation

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

Today, we are going to talk about translation.0002

As an overview, first thing we need talk about is how we can link transcription to translation.0007

After that, we will just give an overview of translation and going to it all the way through.0013

First, we will talk about ribosomes and their ribosomal subunits.0018

Then, we will go through each part of translation, starting with the charging of tRNA,0022

all the way through to initiation, elongation, and termination of translation.0027

Finally, we will briefly talk about the consequences of altering the genetic code.0033

Transcription, once again, to remind us from last time, is making RNA from DNA.0042

We synthesize RNA complementary to DNA.0052

This is going to occur in the nucleus; this is important to remember.0059

Translation is the process of synthesizing a polypeptide from an mRNA transcript.0069

It synthesize polypeptide from mRNA transcript.0080

If we have our tRNAs, our rRNAs, those are not going to be made in a protein.0084

You are not going to translate those RNAs.0089

Obviously, our non-coding RNAs, you will not translate either.0092

Our mRNA transcripts are the ones that are going to go through translation very often.0096

We have our mRNA being translated into 5 prime to 3 prime direction,0104

just as if we were talking about DNA synthesis happening in a 5 prime to 3 prime.0109

Meaning, the next base is added to the 3 prime end.0114

mRNA is translated 5 prime to 3 prime, 3 bases at a time.0119

This is important to know, 3 bases at a time which we call a codon.0125

Each 3 base pair unit or each 3 base unit on an mRNA is called a codon.0134

Each one of those codons will code for a specific amino acid or a stop.0143

The mRNA gets translated 3 bases at a time, by base pairing with a tRNA, having a complementary anti-codon.0150

Your codon and your anti-codon will match up via a base pair.0164

An example being, let us say UAG, your codon, your mRNA, would match up with AUC on your tRNA.0171

Remember, we would have a hydrogen bond between there.0194

Translation is going to occur in the cytoplasm.0202

It will either occur inside ribosomes that are located on the rough endoplasmic reticulum0209

which the endoplasmic reticulum is studded with ribosomes, that is why it is called the rough endoplasmic reticulum.0214

We also have the smooth ER.0223

It is going to be either in the rough ER or in the cytocell itself.0225

If we look at this depiction down here, we can see that here is our nucleus.0231

We have transcription occurring in the nucleus, DNA being made into RNA.0240

In this case, we are looking at mRNA.0245

That mRNA can leave the nucleus into the cytoplasm.0248

At which point are right here, this is our ribosomes and it is going in this direction.0253

Our ribosome can be the site of protein synthesis by reading the mRNA,0261

one codon at a time and recruiting our tRNAs to come in and0268

add its individual amino acid to the growing polypeptide chain shown here.0277

We will go over each one of these steps of translation today and0284

hopefully we will have a very good understanding, by the end of this unit.0289

As an overview, what is happening in translation?0296

First things first, we have a ribosome binding to an mRNA molecule.0299

It is going to bind to it and it is going to search for the start codon.0308

A start codon, this is going to be found on the mRNA.0314

Your start codon is going to be AUG.0318

Remember, this is on the mRNA.0323

This AUG will code for methionine.0327

Inside the ribosome, charged tRNAs, that means they are carrying an amino acid,0337

will base pair to the mRNA via the complementarity found between the anti-codon on that tRNA and the codon on the mRNA.0345

This is important, codon on the mRNA and anti-codon on the tRNA.0357

The next step is, we have the amino acids being transferred from the tRNA and0368

being linked to the growing polypeptide chain via a peptide bond otherwise known as an MI bond.0373

This growing polypeptide is still being associated with the ribosome itself.0384

Spent tRNA, tRNAs that no longer have an amino acid because it has already given its amino acid and0391

let it attach to the growing polypeptide chain, the spent tRNAs get released from the ribosome via the E site or the exit site.0398

We will talk about that later.0410

The process of bringing in a new tRNA having a peptide bond being synthesized and0414

a growing polypeptide continuing in its synthesis, we will continue until you reach a stop codon.0422

Once again, remember it is a codon, it would be found on the mRNA.0430

Our stop codons are UAA, UGA, and UAG.0435

We continue until we reach a stop codon.0449

What will happen is instead of a tRNA coming in, we have a protein called a release factor coming in.0451

What that does is, it basically hydrolizes or cleaves the polypeptide,0459

releasing it from the ribosome and allowing it to fold into its final tertiary shape.0465

Those are our main steps, that is our overview.0473

Let us first start with what ribosomes are and what the subunits are.0477

First of all, a ribosome is a nucleoprotein.0483

It is made up of several proteins, as well as ribosomal rRNA.0489

Remember, our rRNAs + many proteins will make up our ribosome.0494

We have different ribosomes in eukaryotes and prokaryotes, but they serve basically the same function to synthesize proteins.0513

Let us first look at our prokaryotes.0522

Our prokaryotes, the overall ribosome, it is a same thing as if we were looking down here, it is a 70S.0524

What that is, an S is a density unit, it is a Svedberg unit.0533

We have the small subunit which would be down here being a 30S small subunit.0542

The large subunit which is the top part, the bigger part, would be the 50S subunit.0550

In our prokaryotes, the rRNA that is included in the 30S or the small subunit is our 16S rRNA, as well as several proteins.0559

Our 50S contains the 5S and 23S rRNA, as well as our protein subunits.0570

It is important we have this 23S rRNA and this is our ribozyme.0579

It is our peptidyl transferase.0585

In eukaryotes, we have what is called an 80S ribosome, it is a little more dense.0603

We have our small subunit including our 18S rRNAs, as well as proteins and that is called the 40S subunit.0609

We have our large subunit being the 60S subunit and that contains 3 different rRNAs, our 5S, our 58S, and our 28S rRNA.0618

Our 28S rRNA is also the ribozyme found in the eukaryotic ones.0628

That is the peptidyl transferase enzyme in eukaryotes.0637

Each ribosome fully functional, full unit ribosome, our 70S and 80S.0642

Each complete ribosome has 3 different very important sites.0649

We have the A-site.0654

The A-site which can be found right here, is known as the aminoacyl site or the acceptor site.0656

We have that P site which is known as the peptidyl site, the peptidyl tRNA site.0665

This is where we are actually making the new peptide bond.0670

The A-site is where the new tRNA will come in.0675

And then, we have the E-site which is the emptier exit site and that would be not only here,0678

but this would be the E-site right here.0684

That is where the spent tRNAs are leaving the ribosome, so they are being released.0687

Let us talk about the major steps of translation again.0697

First of all, we have the charging of the tRNA, followed by initiation, elongation, and termination.0700

In activation or charging, the proper amino acid is esterrified to the tRNA by its carboxyl group to the 3 prime OH of the tRNA.0707

Remember, a bond between a carboxylic acid and an alcohol makes an ester.0724

If you remember, our tRNA, our cloverleaf structure, we have the CCA at the 3 prime end.0738

That A is where we are going to esterify the amino acid.0752

Let us say for example, this would be methionine.0758

Methionine would be added right there.0763

Next, we would have initiation.0768

Initiation would involve the small subunit of the ribosome binding to the 5 prime end of mRNA,0770

with the help of initiation factors which we will abbreviate as IF.0781

The next step would be elongation and that is going to happen when we have the next aminoacyl tRNA.0789

This is the charged tRNA.0797

When the next one comes in, bind to the ribosome along with the GTP because you need a lot of energy for this to occur.0801

When we have energy and a charged tRNA coming in, as well as elongation factor, we can allow elongation to occur.0813

We are lengthening the polypeptide, longer and longer.0823

Finally, we have termination occurring, when we have the acceptor site of the ribosome encountering a stop codon on the mRNA.0827

Remember, our stop codons are UAA, UAG, and UGA.0838

When this happens, we have the releasing factor.0846

If it is eukaryotes, it is ERF1.0850

If it is prokaryotes, it is just RF1 or RF2.0854

And that will cause the release of the polypeptide chain from the ribosome because it is hydrolyzing it and basically, releasing it.0859

We will get more into the release factors, as we hit the termination section in this presentation.0867

Let us start with the first step, the charging of our tRNA.0876

We have an enzyme called aminoacyl tRNA synthetase.0880

And, that is going to catalyze a two step reaction0887

that results in the covalent attachment of the carboxyl group of an amino acid to the ribose of the adenosine of the CCA.0891

This is, let us say CCA.0913

We have the carboxyl group of an amino acid.0919

Let us say we have methionine.0925

We are going to say its carboxyl group.0928

Each carboxyl group is going to attach via an ester bond to the adenosine on this 3 prime end.0933

This is 3 prime, this would be 5 prime.0946

Once again, this is a tRNA.0951

We have an ester bond.0956

We have our covalent attachment.0968

Right here, it is an ester bond between the ribose of the adenosine0973

found on the 3 prime end of the tRNA and the carboxyl group, in this case methionine but it can be any amino acid.0979

How is this occurring?0989

It is a two step reaction, we are not going to get in all the detail.0992

We have that esterification.0996

We have two different types of aminoacyl tRNA synthetase enzymes.0998

We have class 1 and that attaches the amino acid to the 2 prime hydroxyl group of the ribose.1002

We have class 2 that attaches the amino acid to the 3 prime hydroxyl of the ribose.1012

The end result is the same, we have an ester bond between our tRNA and the amino acid, it is just where it is occurring.1019

What is very important about this reaction is that it is highly specific.1030

The reason that it is highly specific is because, let us say this anti-codon, it has to be specific, hopefully, for the methionine.1038

This would be specific for the methionine, whatever that 3 base pair sequence would be.1049

If for some reason, we have a different amino acid, the methionine being attached to the specific tRNA.1055

What will happen is, when this tRNA properly base pairs with our mRNA,1066

which has the codon for the methionine, this is supposed to hydrogen bond together.1072

If this amino acid is not methionine, we are going to add the improper amino acid to the growing polypeptide chain,1079

even though we have proper base pairing between the codon and anti-codon.1090

This, the whole reason that this is a highly specific reaction is that we have high fidelity of our translation.1095

Because if we start adding in amino acids that are not supposed to be attached to the specific tRNA according to the anti-codon,1101

then we are going to mess up that protein and could really detrimentally hurt that cell.1114

The last thing I want you to know about our charging is that we have ATP energy being required.1122

Once we have charged our tRNA, we can continue on with initiation.1129

Let us start with our prokaryotes, our bacteria and virus section.1137

Let us look at it, first of all, we have initiation factor 3 binding the small subunit, the 30S, at E-site, the exit site.1145

The whole reason that it does this is so that the 50S subunit cannot bind.1160

If we have the exit site here, we have IF3 binding and that does not allow the full 70S ribosome complex to be constructed.1174

The IF3 protein binds the 30S subunit after the previous round of translation is finished, so that you cannot rebind.1205

Next, we have IF1 binding the 30S subunit at the A-site.1215

This would be the P and that would be the A.1229

We have IF1 and that prevents any tRNAs from binding because the tRNAs are going to come in at the A-site.1233

Next, we have IF2 and GTP binding IF1.1249

And then finally, we have our 30S, the small subunit, let us draw that.1265

This is the 50S, this is the 30S.1271

Our 30S subunit will then associate with the mRNA.1277

It will do that by binding to what is called the Shine-Delgarno sequence which has a consensus sequence of AGG, AGG.1281

It is near the 5 prime end of the mRNA.1292

The mRNA, let us draw it in purple.1296

The mRNA will bind to the 30S subunit using its Shine-Delgarno sequence.1304

The Shine-Delgarno sequent has homology to the 16S rRNA that is found in the 30S subunit.1333

We have the 16S rRNA.1344

Once that happens, we have a special tRNA coming in.1359

We have a tRNA that is carrying an N formylated methionine.1367

By the way guys, what a formyl group, it is just a C double bond OH.1374

When it is bound to an R group, we just call it an aldehyde.1382

It is a formylated methionine, it has this group attached to it, binds to the mRNA at the proper start site, the AUG.1396

That is facilitated by our IF2 and GTP that is already there.1408

That helps position the start codon in the P-site.1414

The P-site is where we are going to have the peptidyl transferase reaction.1418

That is where we are actually going to catalyze the synthesis of that peptide or MI bond.1423

At this point, once we have this tRNA, this formylated methionine tRNA bound in there,1429

we have a complete 30S initiation complex.1437

After this occurs, IF3 will be released letting the 50S subunit bind.1446

Remember, IF3 was the one that did not allow the 50S subunit to bind, after the previous round of translation.1454

What happens next, we have IF1 and IF2 being released.1464

Now, we have a fully formed 70S initiation complex that has both an empty A-site and an empty E-site.1468

We have this N formylated methionine tRNA in the P-site.1477

This formylated methionine, we do not have to worry too much about1486

because we have an enzyme called deformylase that removes these formyl group from that tRNA,1489

either during or after translation.1500

We do not see the formylated version in the mature protein.1502

Let us draw out this prokaryotic initiation complexes.1512

First off, let us draw out the 30S initiation complex.1518

First off, we have a 30S ribosomal subunit.1542

We have the E-site, the P-site, the A-site.1560

We are going to have our IF3 protein binding the E-site and that make sure that the 50S subunit cannot associate with the 30S.1569

We then have IF1 binding the A-site.1591

We also have IF2 and GTP binding IF1.1600

We will then have the mRNA coming in, binding the ribosome via homology to the 16S rRNA.1612

The 16S rRNA is binding to the Shine-Delgarno sequence found on the mRNA.1632

Finally, we will have that formylated tRNA coming in and binding to the P-site.1639

We will say this is, we will just call it met-I for the initiation.1652

It is a formylated one, it is a special one.1668

That is our 30S initiation complex.1672

Let us look at our 70S initiation complex.1675

Remember, S is a Svedberg, it is a density unit.1687

What we have now is our 30S, it still has its exit site, its peptidyl site, and its acceptor site.1697

We have the mRNA properly in there.1711

We have lost IF3, now we can have our 50S subunit binding.1720

IF1 and IF2 have left, as well.1736

We still have our original tRNA with that initiation special methionine in there.1739

Remember, the anti-codon of the tRNA is going to base pair complementary with the codon on the mRNA.1756

This is our 70S initiation complex.1771

What about translation initiation in eukaryotes?1778

Same concept, a little more complicated.1782

In eukaryotes, the small subunit, our 40S, is actually already associated with the initiation tRNA, before it is recruited to the mRNA.1787

And then what we do, we will form what is called a 43S pre-initiation complex.1802

What that consists of is the following.1811

We have the 40S subunit, the ribosomal subunit.1815

That is going to bind to the 5 prime cap of the eukaryotic mRNA.1818

Remember, the eukaryotic mRNA has the 5 prime cap.1823

What we have after the 40S subunit binding to the cap, we have eukaryotic initiation factor 1 binding to the E site.1829

We have EIF5 binding to EIF1.1839

EIF1-A binding to the A-site.1843

EIF3 binding to that one, EIF1-A.1846

And then, EIF2 and GTP binding the initiator methionine tRNA and recruiting that to the peptidyl site, the P-site.1850

Part of eukaryotic translation initiation is the circularization of the mRNA.1868

This occurs via a complex called EIF4.1875

It is EIF4-A, EIF4-G, EIF4-E.1880

EIF4-A will bind to the upstream region of mRNA.1885

Let us draw this up, as we are talking about it.1890

First off, we have the mRNA that we are going to draw.1895

We will denote that it has a cap right here.1903

EIF4-A will bind to an upstream region on mRNA.1911

Let us say, here is A.1921

EIF4-G binds to the poly-A tail via hydrogen bonding.1928

EIF4-E binds to the 5 prime cap.1940

What this allows is for, let us say where the start codon is.1949

Remember, this is all upstream.1956

Let us say the start codon is right there.1958

Here is your start.1961

Remember, our start codon is AUG.1965

AUG codes for methionine.1968

What we can have by circularizing this, we actually allow ribosomes to bind in several different places.1978

Small subunit, large subunit, all the way up until our stop codon.2011

Our stop codons are UAG, UGA, or UGG.2031

Sorry, not UGG, UAA.2055

UAG, UGA, UAA.2059

These do not code for a tRNA, and therefore, do not code for an amino acid.2065

It codes for a release factor.2072

Each of these ribosomes can bind to the circularized mRNA, making what we call a polyzome.2081

What we will have is a little bit of protein.2094

As it goes further along, it will get longer and longer.2100

These are all different ones.2105

These are all proteins.2112

Those are all being made, being synthesized, translation is occurring at that time.2122

What we are doing is we are making several different proteins or probably the exact same protein.2126

We are making several copies of the protein from a single mRNA.2133

If we have multiple ribosomes translating, we are getting multiple copies of the protein from a single mRNA.2138

We talked about the 43S initiation complex.2151

Let us move to the 48S pre-initiation complex.2155

The 48S pre-initiation complex is the 43S.2160

We talked about plus the addition of the circularized mRNA with the EIF4-E, G, and A subunits.2165

The 48S pre-initiation complex will scan the mRNA, 5 prime to 3prime, looking for the AUG with the correct reading frame.2178

The AUG, meaning it is looking for the start codon in methionine.2187

It is looking for in the correct reading frame.2193

At the very end of this unit, we will talk about reading frames.2195

For right now, let us just move on a little bit.2199

This right here, looking for the AUG in the reading frame is an ATP dependent process, it is energy using.2205

It is facilitated by EIF4-4A which actually has helicase activity and EIF4-B.2213

EIF4-B actually activates EIF4-A.2219

Once the proper AUG is found, our initiation factors 1, 2, and 5 release which allows EIF4-B and GTP to bind that initiator tRNA.2229

We lose a bunch of initiation factors and gain one which helps a tRNA bind.2246

This then, recruits the large subunit to associate making the fully functional 80S ribosome,2255

with the proper initiator methionine tRNA loaded into the peptidyl site.2266

Similar to the prokaryotic initiation, we have all the initiation factors get everything ready.2274

Then, they leave so that you can have the full association of the large and small subunits,2282

with the proper initiator tRNA in the peptidyl site.2289

Just as a reminder, we have the cap or if we do not have a cap which is actually fairly common on mRNAs in eukaryotes.2296

If you do not have a cap, you utilize what is called the kozak sequence with the consensus sequence ACC, AUGG.2305

Whether it is the cap or the kozak sequence that will help the 40S ribosomal subunit bind to the mRNA.2315

And then, the 60S subunit will bind at the AUG, as we talked about up here once it has been found.2325

Let us show what this would look like.2335

We have our 43S pre-initiation complex.2338

Here we go, we have our 40S ribosomal subunit with the E, the P, and the A-site.2362

We have the EIF1 binding here.2379

This does not allow the 60S subunit combine.2388

We have EIF5 binding this.2394

Over here at the A-site, we have EIF1-A binding here, that is blocking the acceptor sites so that we can have new tRNAs coming in.2403

And then, we have EIF3 binding that.2416

We have our tRNA coming in here.2426

This is going to be attached to that specific initiation methionine.2442

And then, we have our last one, we have EIF2 + GTP bind in here.2447

That is going to help position the methionine in the proper P-site.2470

This is the 43S.2474

Now, if we talk about our mRNA, let us just say here we have our mRNA, our cap here.2477

I’m going to draw in a non-circularized form just so we can see it easier.2503

We have EIF4-E down to the cap.2509

We have EIF4-G bound.2518

We have the EIF4-A helicase.2527

And then, we have EIF4-B which activates this helicase bound right here.2535

This one + this one equals our 48S pre-initiation complex.2544

Just so we can see one more, let us write out what happens when we have the full association.2574

We have our 40S, we have our E, our P, our A.2582

Once we have our initiation factors leaving, that allows us to have association with the large subunit, the 60S.2589

And then, we have that mRNA coming through.2600

We have our proper initiation tRNA in the P-site.2610

We will have an empty E-site and an empty A-site.2620

And then, we now have the full 80S complex.2623

And now, it is ready to go through elongation.2630

The next step, elongation.2642

This is going to just repeat over and over again.2646

Elongation, every time a new tRNA comes in, this is the step that is going.2649

We have charging happening to every tRNA.2655

We have initiation happening once.2659

We have elongation happening for every tRNA that is coming in.2661

We have termination happening once.2665

We have our incoming charged tRNA, aminoacyl tRNA, being recruited by, if were eukaryotes,2670

it is EF1-α, elongation factor 1-α.2678

For prokaryotes, elongation factor-T.2685

The incoming charged tRNA binds the complementary codon on the mRNA in the A site.2690

Remember, it is using its anti-codon to bind the codon.2699

Next, we have peptide bond formation being catalyzed by the enzyme peptidyl transferase.2705

Remember that is a ribozyme, either the 23S rRNA, if it is prokaryotes.2714

Or the 28S rRNA, if it is eukaryotes.2723

The peptide bond formation occurs between the amino acid of the tRNA that is in the A site, it just came in.2730

And, the C terminal amino acid of the growing polypeptide chain which is attached to the tRNA that is already in the P-site.2740

I will draw this out in just a couple of slides.2750

Finally, the growing polypeptide chain gets transferred to the tRNA in the A-site.2755

We will have what is called translocation occurring.2761

As I said, translocation occurs and that is enabled by the action of elongation factor 2, if it is eukaryote, EFG, if it is prokaryote.2767

Translocation moves the tRNA in the P-site.2781

Now, it does not have amino acid anymore.2785

It moves it to the E-site.2788

Remember, the polypeptide chain has been transferred already from the tRNA in the P-site to the tRNA in the A-site.2790

Now, we do a shift.2799

The tRNA that was in that A-site, the one that just came in,2801

now it is attached to the growing polypeptide chain will move to the P-site.2805

The tRNA that was in the E-site, the exit site, is released.2812

And, the next charged amino acid which is dictated by the codons in the mRNA will enter into the A-site.2817

Let us draw this out so that we know what it looks like.2828

I will draw my ribosomes in blue.2836

I will label this for us.2846

We have 40S and we have 60S.2848

We have the E-site, the P-site, the A-site.2853

This is our mRNA and it is the 5 prime end.2872

We are going to redraw this bigger.2883

We have the E, the P, the A.2926

We have going to have the same channels in the 60S as well.2930

Our mRNA, this is 5 prime mRNA.2939

At first, I’m just going to very simplify what a tRNA looks like.2946

I’m just going to draw it just like this, attached to, let us say a particular amino acid.2954

That is what I’m going to say an amino acid looks like.2964

This is perfectly what we have for initiation.2967

The next step, as the mRNA stays in one place, the ribosome will actually shift to the right.2974

We will now go to the next codon.2987

What we have is or what the next step would be, would be a new charged tRNA coming in2990

with a different amino acid, let us say.3007

What we have is called translocation.3015

As the ribosome moves, what we end up having is a kind of a shift looking like this,3067

whereas, the tRNA are still bound in the P and A site.3080

However, they have shifted looking like this.3085

What we are actually doing in that P site, we are making that peptide bond, if I move it.3088

What we have done is we have transferred this amino acid, the methionine, to this tRNA.3115

We have made a peptide bond or MI bond.3127

Now, we have a growing chain.3131

As we can see, the first amino acid was the methionine, the blue.3133

The second amino acid, it could be whatever you want to call it.3138

Let us just say it is alanine.3141

We have had that translocation.3144

The next step would be the full movement to where it looks like this, 40S, 60S, the E, P, A, E, P, A, the 5 prime mRNA.3146

We have a full movement, as we are translocating.3177

We have the first tRNA, now without an amino acid attached to it.3198

It is moved into the E-site.3205

The last tRNA that has come into the A-site, now has 2 amino acids attached to it.3207

The growing polypeptide chain is now fully in the P-site.3217

Now that opens up the A-site for the next tRNA to come in.3222

Let us see what that looks like, to continue this way.3227

What we have here, it is going out already.3259

We now have this one here, that is exited.3269

We have a new tRNA coming in with another amino acid.3284

The whole thing will occur all over again.3299

You can just look, what would happen next would be, this would be the next step.3301

It would follow to here and then back to here again.3310

That would continue on over and over again until we hit a stop codon, in which case then we will go through termination.3315

Now that we are talking about termination, we need to talk about release factors.3327

These are proteins that will terminate translation, when the ribosome comes to a stop codon.3333

Remember, stop codon is UAA, UAG, UGA.3340

The release factors are proteins, they are not tRNAs.3350

They do not carry an amino acid.3353

This is very important to understand.3355

It is so important that I’m just going to highlight it all.3359

I’m even going to put a red star by it.3367

There are two classes of release factors.3373

We have class 1 release factors.3376

They will recognize stop codons and hydrolize or cleave the polypeptide chain from the tRNA that is found in the P-site.3381

We have, if we are talking about prokaryotes, these are prokaryotic release factors.3393

Release factor 1 recognizes UAA and UAG.3405

Release factor 2 recognizes UAA and UGA.3410

Eukaryotic release factor 1 will recognize all 3 stop codons.3416

Class 2 release factors will help release class 1 release factors from the ribosomes.3423

Class 1 release factors cleave the chain from the tRNA in the P-site.3431

It allows the protein to leave the ribosome.3439

Class 2 release factors release the class 1 release factors from the ribosome.3442

The class 3 release factors are RF3 in prokaryotes or ERF3 in eukaryotes.3450

I put together a little table so that it might be easier for you guys to understand and have all the information in one place.3462

This should just say step.3474

Your tRNA charging, the whole function of that is adding an amino acid to the tRNA.3481

The protein or the enzyme that is responsible for that is aminoacyl tRNA synthetase.3487

One thing I want to point out guys, synthetase, a general rule of thumb,3495

when you see the word synthetase that means it is an enzyme that is making something, synthesizing.3499

It needs the input of energy, usually ATP or GTP is needed.3508

If this were synthase, it is an enzyme that is doing a synthesis reaction but does not need the input of energy.3516

That is just a biochemical aside.3528

You will learn that in biochemistry.3530

The next step of translation is initiation.3533

The recognition of the start site, for eukaryotes that is done by EIF1-A and EIF3.3537

For prokaryotes, it is IF3.3545

Recruiting the special initiator methionine tRNA to the P-site, eukaryotes, that is EIF2.3549

Prokaryotes, that is IF1 and IF2.3556

For elongation, recruiting all other tRNAs to the A-site, prokaryotes, it is EF1-α, for prokaryotes, EF2, as TU.3561

For peptide bond formation, whether it is eukaryotic or prokaryotic, the name of that enzyme is peptidyl transferase.3577

It is a ribozyme, meaning it is a ribonucleic acid that has enzymatic activity.3585

For you eukaryotes, that is the 28S rRNA and prokaryotes that is the 23S rRNA.3593

Either one of those are found in the large subunit.3600

Eukaryotes, that 28S rRNA is found in the 60S subunit.3603

Prokaryotes, the 23S rRNA is found in the 50S subunit.3608

Finally, translocation involved in the elongation, that is EF2 for eukaryotes, EFG for prokaryotes.3619

You are going to need GTP, for each time it translocates.3627

It is energy requiring.3632

Finally, termination, this is the recognition of stop codons and the release of the polypeptide chain, that is done with our class 1.3635

The hydrolization of the polypeptide chain from the tRNA in the P-site,3646

it is done by class 1 release factors which is ERF1.3653

If we are talking prokaryotes, RF1 and RF2.3660

The class 2 release factors which released the class 1 release factors from the ribosomes is ERF3 for eukaryotes and RF3 for prokaryotes.3663

Let us do a quick review of translation.3677

Each tRNA with its own anti-codon is attached to a specific amino acid, via the CCA end on the 3 prime.3680

The amino acids delivered by the tRNAs are joined together to form a polypeptide inside the ribosome.3689

Remember, the tRNA is utilize the complementary of its anti-codon to the codon on the mRNA.3696

The ribosome will move along the mRNA and encountered codons3704

which are 3 base sequences that will tell whatever tRNA to come in and add a specific amino acid.3710

Multiple ribosomes can translate the same mRNA at the same time, creating a polyzome which I talked about when I circularized the mRNA.3717

The next thing that happens, we have a charged tRNA entering the A-site, where the anti-codon hydrogen bonds to the codon.3728

Next, the tRNA translocates to the P-site.3738

The attached amino acid gets connected to the previous amino acid of the growing polypeptide chain.3743

And then, the spent tRNA, the one that no longer has amino acid on it, exits from the E-site.3749

It continues over and over again until a stop codon is reached.3755

I briefly said that I wanted talk about consequences of altering the genetic code.3764

We have just a couple slides and I want to explain to you why this is so important.3769

Our genetic code, if we look back at our codon table which I have on the next slide.3775

Each 3 unit or each 3 base sequence which we call a codon, codes for a specific amino acid.3780

Each one of those amino acids are in a protein, for a specific reason.3788

Maybe they are supposed to make hydrophobic bonds or hydrophilic bonds, ionic bonds, so on and so forth.3794

If you alter that sequence, if you are adding in an amino acid that should not be there,3801

you can affect the overall function of the proteins.3808

There are couple different types of mutations that I want to talk about.3813

We have first, what are called silent mutations.3817

These are the types of mutations that we can call no harm, no foul.3821

These are mutations in the DNA sequence that do not alter the amino acid sequent3825

due to our degeneracy of the genetic code.3832

Example, all 4 of these and actually 2 other triplets, all 4 of these code for serine.3835

If we happen to, instead of what should have been a UCU.3843

If for some reason, we have a mutation changing the U to a C, or even an A or a G, no harm no foul, we still get a serine.3848

It is a silent mutation, we do not even see it.3860

If we get what is called a missense mutation, that is a mutation in the DNA sequence3864

that leads to a change in the predicted amino acid sequent.3871

For example, instead of a UGC that is supposed to be added, there is some sort of mutation and3875

we actually have a G present, instead of a C in that 3rd triplet base.3883

That changes the cystine to a tryptophan.3888

Maybe, we were supposed to be making a disulfide bond using our cystines in the protein.3892

But now, we no longer have that ability, so that can be detrimental.3897

Missense mutations do not always have to be problematic because, let us say, we have aspartate and it is change to a glutamate.3902

You still have a negatively charged amino acid.3911

Maybe, it can participate in the same exact bond that it was supposed to have made.3915

Missense mutations do not always have to be showing a type of phenotype but they can.3921

The last one, the most detrimental one, is nonsense mutation.3928

That is a mutation in the DNA that changes a normal amino acid codon to a stop codon.3933

This truncates the protein, it makes its smaller.3942

An example, we have UAU coding for tyrosine.3948

All we have to do is have that one base changed to UAA being a stop codon.3953

Maybe, instead of getting a full 6,000 amino acid protein, maybe we only get 60 amino acid protein.3958

If that is the case, it is not going to be very functional and we are likely going to degrade it and just throw it away.3974

What can cause even more problems is if, let us say, we get a protein that can still function but it functions in a different way.3980

And thereby, maybe causing a genetic defect.3993

Remember that the consequence of altering the genetic code are due to the possibility of changing the triplet code.3997

Cystine, it is fine, if you change that last one, the U to a C, not a big problem.4007

But if you change that U or C to an A, that is going to give you a stop codon.4012

That is an example of a nonsense.4021

If you change the U to a C right here, that is an example of a silent because it is still going to be cystine.4026

If you change the U to a G, that is going to give you a different protein or a different amino acid.4034

That is an example of a missense.4040

Between these two silent, between these two nonsense, between these two missense.4045

I want to end with just an example of what a frame shift mutation is, because this can alter the genetic code.4064

Frame shift mutation is due to the addition or deletion of nucleotides, in other than multiples of 3.4074

And, that will shift the open reading frame which most often results in truncated protein.4081

Let us write out a sequence, ACG, this is the mRNA.4087

UGU, ACA, UGG, AUG, CUG, GUAA.4103

There are 3 possible reading frames for an mRNA and that is due to the fact that there are 3 bases per codon.4127

For example, this could be your open reading frame 1.4135

Red is ORF1, we can have purple being the next reading frame, starting there, that is ORF2.4141

Let us say green is the next one, ORF3.4154

That is all we can have because, if we then go to starting at the next one, this UGU, that is still ORF1.4163

This is important, we have 3 different open reading frames.4173

If we search down here, we can find that there is an AUG.4177

We have an AUG that we can find here and AUG that we find here.4183

Those are start codon.4186

Then, we can find one UAA, this is a stop codon.4189

If we look, we can see that if we go red, 33333, this is actually a start codon only for open reading frame 1.4196

If we look for open reading frame 2, if we go 3333333, this is a stop codon only for open reading frame 2.4228

If we look over here, open reading frame 3, this is 333, this is a start codon only for open reading frame 2.4260

This should be in green.4293

This is important because, let us say for example, we are looking at our 3 possible open reading frames.4308

We see two different starts and one stop.4321

Let us say, for example, that we are going to do our translation through open reading frame 3.4324

We can start here, we have AUG, that is going to be our start, our methionine.4333

Then, we will have GAU as our next codon.4338

Then, GCU, then UA, whatever that next one is.4342

We will write it in as, let us say A.4349

All the starts and stops are perfect as they are now.4359

But if we would have a frame shift mutation, either adding in or subtracting bases other day in 3,4363

you either add or subtract 1 or to 2 bases, that can affect the open reading frame.4372

If we have in addition of a single base, and if we say we add it right here, frame shift caused by the addition of an A right here.4378

What that does is, it shifts the open reading frame.4395

The new one, I’m going to write it in red.4398

This would be AUG, GAU, GCU, GCA, UAAA.4402

What we see here, this is open reading frame 3.4418

What we see is we have a start codon, an amino acid, an amino acid, an amino acid, a stop codon.4426

This is a truncated protein now.4434

Instead of going on from maybe 500, 600 thousands of amino acids,4436

this now is a 4 amino acid polypeptide, once it hits that stop codon.4442

Likely, this will just be sent to the cellular trash can, the prodiuzome or the lysosome,4453

and be broken down, degrade it, and you have to restart.4460

This is how a frame shift can affect the genetic code because this might not be as big of a problem,4463

if we can overcome the frame shift.4472

But if not, then maybe this protein is not made at all in the body in its functional form and4474

that could possibly lead to a disease or cancer, or something.4481

To where, maybe it is a lethal type of mutation and there is not even life form from it.4485

That is the end of our unit today, thank you for joining us at www.educator.com. I hope to see you again next week.4494