Understanding DNA Transcription: A Comprehensive Guide

what's up ninja nerds in this video today we are going to be talking about dna

transcription before we get started if you guys do like this video please hit that like button comment down in the

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facebook instagram patreon all that stuff will be there all right ninjas let's get into it all right ninja

so with dna transcription we have to have a basic understanding of just the definition what the heck is

transcription and it's really a simple thing it's just taking dna

okay double stranded dna with the eukaryotic cells and even in prokaryotic cells

and converting that into rna so it's taking dna and making rna that's all transcription is

but in order for transcription to occur in order for it to take place we need two particular types of proteins

or enzymes if you will to facilitate this process and i want to talk about those real quick

because these are very important now transcription can be kind of different okay and it's important to know the

differences between prokaryotic cells we'll consider bacteria in this case

and eukaryotic cells human cells like me and you in prokaryotic cells there's a

particular type of protein that is needed in order for transcription to take place

what is that protein so let's say that we take this dna strand here right we have this

dna strand on this dna strand we have these blue portions that i've highlighted here as a

box with some lines in it this right here for right now i want you to know is what's called a promoter so

this is called a promoter region now a promoter region is a

particular nucleotide sequence within the dna and what it does is it allows for

particular proteins like rna polymerases and transcription factors

to bind onto the dna and then start moving through the dna taking the dna and making rna so that's

the first thing you need to know is within the dna there's a particular nucleotide sequence

we'll talk about a little bit later called a promoter region and that's the first thing that we need to

identify let's say that we take this particularly for prokaryotic cells so prokaryotic cells

and we'll just say like a bacterial cell okay prokaryotic cells use a very particular type of enzyme what

is that enzyme it's called a rna polymerase holoenzyme okay so it's called an

rna we're going to put poll polymerase holo enzyme now that's a lot of stuff let me explain

what this is and i'll show you the structure of it a basic structure of what the rna polymerase holoenzyme is

it's made up of two things one of the components of this enzyme is called the core enzyme

and the core enzyme for this rna polymerase holoenzyme consists of multiple subunits that they

just love to ask you on your us mles and other exams and these are they contain two alpha

units okay two alpha chains proteins it contains

two beta units technically we say beta and beta prime if you really want to be specific and

then one more which is called an omega unit okay so these are the primary components of

the core enzyme which makes up rna polymerase what's important to remember is that these are what are going to

really read the dna and make rna that portion of the enzyme reads the dna

and makes rna the next component of the rna polymerase holoenzyme is the portion that we need in order to bind

to the dna to the promoter region without it we won't be able to allow for this rna polymerase to bind to the dna

and transcribe it this is called the sigma right or you can represent it like this

subunit or factor if you will okay these two components the core enzyme which is made up of the two alpha the

beta and the beta prime and the omega sub unit as well as the sigma subunit make up the

entire rna polymerase now let me show you for example here let's say i represent the core enzyme

as just this kind of blue circle with lines in it and then we'll represent the sigma

subunit as kind of like a pink circle with some lines in it right so let's imagine here

we have that core enzyme which we're going to represent like this and then the other component of it which

is the sigma subunit which will represent like this that sigma subunit will then

bind to the promoter region once it binds to the promoter region then this core enzyme of the rna

polymerase will then release away from the sigma subunit and it'll start moving down

this dna and as it moves down the dna it'll read the dna from three to five and synthesize an rna

strand from that which we'll talk about more detail later

from five to three so it'll read the dna and make rna this rna that we make from in

prokaryotic cells with the rna polymerase holoenzyme is very different from eukaryotic cells

in prokaryotic cells that mrna that we made from this one rna polymerase holoenzyme can make all the mr

all the rna we need whether that be rrna within the prokaryotic cell whether that be m

rna within the prokaryotic cell or t rna within the prokaryotic cells so that's very important big thing i

really need you guys to take away from that is prokaryotic cells they use one rna

polymerase which is called a holoenzyme made up of two components a core enzyme made of these subunits

and a sigma subunit the core is what reads the dna and makes the rna the sigma subunit is what

binds the rna polymerase to the promoter region enabling it to transcribe the dna okay

and whenever you make rna within a prokaryotic cell from this rna polymerase it makes

all the rnas within that prokaryotic cell in eukaryotic cells it's a little bit different

so let's talk about that let's say here we have three promoter regions that i want us to

focus on and this is all within eukaryotic cells in eukaryotic cells we need

two different things in order to allow for transcription to occur and this portion here right and this

portion of prokaryotic cells we only need one enzyme which had two different

components within eukaryotic cells each process requires

a particular enzyme an rna polymerase and a transcription factor let's let's kind of write that down

so let's say that we take this first promoter we want to read this gene this portion of the dna

and make rna and this is the rna that we're actually going to synthesize right here okay from this

gene a particular enzyme let's represent this in blue since we've been kind of doing

blue here there's going to be a particular enzyme which is going to read this dna okay

and make this rna there's a particular enzyme what is that enzyme called it's called rna polymerase

but this is the first promoter within the eukaryotic cells that we're talking about right

so let's call it rna polymerase one rna polymerase one will read the dna and make a particular type of rna but in

order for it to do this it needs a special protein that can bind to the promoter region which allows for

the rna polymerase to bind to the dna and read the dna what is that particular protein that

protein let's represent it here in let's do green there's a particular protein

which will bind here to the rna polymerase and to the promoter and allow the rna

polymerase to bind to the dna and start moving down reading the dna and making this rna

what is this called this is called a transcription factor tf and there's many different

types of transcription factors the particular thing that i need you to remember for right now

is that we call these transcription factors which are utilized by rna polymerases

within eukaryotic cells we call these general transcription factors we'll talk about

very specific types with an rna polymerase type 2 a little bit later but for right now

two things i need in order for this rna polymerase to be able to read the dna and make this rna rna polymerase 1 needs

a general transcription factor to bind to the promoter allowing the rna polymerase 1 to

then bind into the dna read it and make rna what type of rna does it make i have all

the rnas within prokaryotic cells from one rna polymerase but rna polymerase 1 makes a very

particular type of rna and this is called r rna now rrna is very important because this

is incorporated into what's called ribosomes ribosomes and ribosomes are utilized in the translation process

where we take mrna and from that make proteins so we'll talk about this later in

another video but for right now first thing i need you know is rna polymerase one

with transcription factors reads the dna and makes rrna now that makes everything else

pretty easy from this point here's another promoter region of a particular sequence of dna

right within a eukaryotic cell so this is the second promoter region another enzyme binds another rna

polymerase and not only just that one rna polymerase here but we also need

a set of general transcription factors to bind to this promoter region so general transcription factors we need to

bind to the promoter region enabling this rna polymerase to bind to the dna

read it and then make what make these particular types of rna we have here this is well this was the first promoter

this is the second one all right so we're going to call this rna polymerase

2 rna polymerase 2 will bind to this promoter via the transcription factor read the dna and make rna what kind of

rna is it going to be making big thing i need you to remember is it's making m

rna mrna you'll see later again is the component it'll have to go through some very specific modifications

that we'll talk about in great detail and then eventually it'll be translated with

the help of rrna and another thing called trna at the ribosomes and making proteins

okay the other thing that you guys can remember if you guys want to be scholarly or ninja nerdy

there's another rna that's made here and we'll talk about it a little bit later with what's called splicing

and these are called small nuclear rnas and these are involved in what's called splicing and we'll get into that a

little bit more in detail later okay but big thing rna polymerase ii with the help of general transcription factors

makes mrna and snrnas rna polymerase one with the help of general transcription factors

makes rnas when the heck do you think this last promoter region of this

sequence of dna within this eukaryotic cell is going to make trnas and it's the same process what do

i need here i need general transcription factors to bind to the promoter region

when that binds that facilitates or it helps to allow the rna polymerase

type what three two bind to the dna and then read the dna

and then synthesize what rna what type of rna is it making the type of rna that is being synthesized from

rna polymerase iii is primarily trna but a teensy little bit of snra is

also made by rna polymerase type 3. and if you guys really want to be extra ninja nerdy technically

even a teensy bit of rna is also made here as well okay now trna what the heck does this do

you'll see later that this is also involved in the translation process

it carries a particular amino acid and an anticodon which is going to be involved in that

process and we'll talk about that in a separate video so i know this was a lot of stuff to

take away and take away from this but the big overall theme that i really just out of

all of this what i want you to take away from this is this quick little thing here

that rna polymerases 1 2 3 remember r m t

rna polymerase 1 primarily gives way to rrna rna polymerase 2 primarily gives way to

mrna and then rna polymerase 3 primarily gives way to

t rna these are the big things that i want you to take away from all this if you want to go the extra mile be extra

ninja nerdy two and 3 also can give way to what small nuclear rna if you really want to

go the extra mile technically three can also give way to rrna but this is the basic thing to take

away from what we just talked about and then the other thing is in prokaryotic cells we don't need all of

these we need one rna polymerase holoenzyme to make all the rnas one last thing is

you notice in eukaryotic cells that we have particular transcription factors that are going to be needed for each rna

polymerase the transcription factor in prokaryotes technically if you want to be specific

is the sigma subunit because it's the portion that's binding to the promoter to allow

the core enzyme of the rna polymerase to read the dna okay so that kind of covers the basic

concepts of the two main things that we need in order for this transcription process

to occur now there's one other thing that i want to talk about very quickly before we really

start talking about mrna because that's going to be the primary topic here i want to have a

quick little discussion on how we can modulate the rate of transcription either

speeding it up or slowing it down okay so the next thing i want to talk about is very very briefly

on eukaryotic gene regulation so i want to have a quick quick tiny little discussion on gene

regulation okay and the only reason i want to mention this is because this is very

easy and it kind of makes sense along with what we're talking about but we're not going to talk about it in

prokaryotic cells we're primarily going to talk about this gene regulation and eukaryotic cells we're going to have

a separate video because it's more involved we'll talk about gene regulation and

prokaryotic cells with the lac operon and the tryptophan operon we'll get into that

but in eukaryotic cells there's two ways that we can modulate and it's really easy

one way that we can modulate transcription is we have particular dna sequences

particular sequences of dna particularly palindromic sequences which are called enhancers and enhancers

are basically dna sequences and the big thing i want you to take away from this they can increase

the transcription rate so they increase the rate of transcription or the process of

transcription okay we'll talk about how they do that the other thing that can regulate the

the transcription process or gene regulation in a way is something called silencers

in silencers they do what they decrease the transcription rate or the transcription process

now it's really straightforward it's relatively simple let me explain what i mean let's say here we have a

strip of dna we're going to explain how this happens so here's our strip of dna and remember this blue region what did

we call this blue region that we talked about above this was called our promoter region and

do you guys remember let's take eukaryotic cells in this case what we needed in order for this process to

occur we needed a particular transcription factor to bind to that promoter region

and then what else did we need in order for that to read the dna and make rna you needed a particular rna polymerase

right so we need an rna polymerase depending on which one we're talking about

would depend on the type of rna that we want to make and then a transcription factor okay

now this is going to go read the dna this rna polymerase will read the dna and then make rna right now here let's

say that we have the promoter and you can have this enhancer upstream from the promoter or it could be down

here downstream where we can't see it in this diagram but it would be all the way down

here regardless of where it is it's usually can be close to the promoter or it can be far to the promoter so you're

probably asking the question how the heck would an enhancer that's really far away

influence a promoter that's all the way down here how that does it do that there's particular structures there's

different things that can activate enhancers and cause conformational changes of the dna

and these are called specific transcription factors you know why i really frustrate i got

really deep into talking about specific and general transcription factors the general transcription factors are

what bind to the promoter region specific transcription factors which we're going to really kind of do a

different color here let's do purple specific transcription factors will bind to this enhancer region so

this let's put specific transcription factors these will bind to the enhancer when they bind

to the enhancer region it causes a looping of the dna to where now the promoter was far

downstream from this enhancer but when this specific transcription factor binds to

the enhancer it causes the dna to loop in a way that it's in very close proximity to the

promoter region even though it's far upstream from it and then what was bound to this promoter

region here do you guys remember the general transcription factors and what else the rna

polymerase so now that these are in close proximity guess what this general trans this uh specific

transcription factor can do to this area over here it can act on these proteins and

stimulate this reading of the dna the rna polymerase is to read the dna and to do what

make rna whether it be mrna rrna trna so the whole point here is that

enhancers can be either far upstream or far downstream which makes it hard to interact with the promoter

but if a specific transcription factor binds to that enhancer it creates a looping process

bringing it in close proximity which can then stimulate the specific transcription factors and the

rna polymerases which are bound to the promoter to increase the transcription of rna

what do you think silencers do the exact same process we're not going to go into detail of it

but if you imagine i did the same thing i put the silencer here and i have a specific transcription

factor that bound here it's going to fold it in a particular way bringing it close to the promoter

inhibiting that promoter region and slowing down the transcription process it doesn't make sense it's pretty cool

too right so i need you guys to ask yourself the questions because we're going to talk about these

these general transcription factors what in the world are these specific transcription factors

and i know that if you guys are the og ninjas you'll know these processes in and out

you guys know when we make a protein whenever we have like a cell signaling response we've talked about

this a million times here an engineer right let's say that we take a hormone like

tsh which stimulates thyroid hormone synthesis right tsh will act on a particular receptor we call these

g-protein-coupled receptors right like g-stimulatory proteins those g-stimulatory proteins will

activate something called cyclic amp cyclic amp will then activate something called

protein kinase a protein kinase a depending upon what type of you know transcription factor you need in this

case we're going to activate a very specific transcription factor

for making what thyroid hormone so some type of thyroid hormone transcription factor

that'll be needed to bind to the enhancer change the shape of it activate the promoter have the rna

polymerase read the gene that makes what hormone thyroid hormone

and so you'd have this get read you'd make an mrna that would then get translated and make

thyroid hormone doesn't that make sense how that process occurs so we can increase the transcription and

protein formation of thyroid hormone through this process the same thing exists

with steroid hormones if i took for example testosterone you guys know testosterone right

testosterone does what testosterone will move across the cell membrane it'll bind onto a

intracellular receptor when testosterone binds onto the intracellular receptor what will that intracellular receptor do

bind to the enhancer when it binds to the enhancer loops it brings it close to the promoter

stimulates the transcription to make proteins within muscle so that you can get direct

right that's the whole process of how we increase transcription and the same thing would

happen if we wanted to decrease it just we would have some type of repressing transcription factor binding

to the silencer that would inhibit the transcription process so i think we have a pretty good

idea now of the basic concepts of eukaryotic gene regulation now spend most of our time talking about

the transcription particularly of mrna all right so when we talk about transcription we've had a basic concept

of it right that we need rna polymerases and transcription factors to read the dna and make the

rna but the real one that i want us to primarily focus on which is primarily important with

transcription of dna is mrna that was the real important one now that's in eukaryotic cells with

utilizing the what rna polymerase type 2 in prokaryotic cells we would just be using the

rna polymerase holoenzyme so what i want us to do is i want us to go through particularly and more d we already have

a basic concept of how this is going to work but let's go into the stages of

transcription particularly for mrna within prokaryotic cells in eukaryotic cells

the first stage that is involved here is called initiation of transcription so the first step that

we have to talk about is called initiation of transcription now this is the part that we've pretty much already

familiarized ourselves with okay now within this let's have our two cells

okay that we're going to do initiation with we're going to have our prokaryotic cells here

on this left side of the board and then over here we're going to have the eukaryotic cells here on the right

side of the board what i want us to do is to have kind of a comparison a side-by-side comparison

of the initiation process the first thing that we need to know is we've talked a little bit about this

already but this blue region what did we call this blue region here again this blue region was called the promoter

region now the promoter region i told you is a particular kind of like nucleotide

sequence that is very very specific and allows transcription factors and rna polymerases to bind to the dna

it's kind of a signal if you will it's like hey here i am come bind to me in prokaryotic cells the promoter

region has particular types of like names and just weird stuff that they can ask you in

your exams so in the prokaryotic cells they call this the third negative 35 region

which means from the start point at which the rna polymerase starts reading the dna and making rna

if you go back 35 nucleotides that's kind of the point at which the rna polymerases will

bind in prokaryotic cells another one is called the negative 10 region

but they wanted to give this one a name so they called it the pribno box just meaning that it's negative it's 10

nucleotides away from that startup transcription right and then the last one here is called the

plus one region which is also called the transcription start site so it's going to be pretty

much the nucleotide at which you just read and start making the whole process of

rna so these are the regions that you guys need to remember within prokaryotic

cells these are the kind of specific promoter regions and eukaryotic cells

the promoter regions have particular nucleotide sequences that we need to be aware of

these are called the top box which means that you would have thymine adenine thymine adenine

that would be a particular recognition sequence within the promoter and eukaryotic cells

or cat c-a-a-t so cytosine adenine adenine thymine and the last one is a gc box

so if there's a tata box a cap box or a gc box these are identify identifying

nucleotide areas at which the rna polymerase is type 2 and transcription factors will bind to

that is the important thing okay now the next thing here is the polymerases the rna polymerase is

within prokaryotic cells it's just one it's rna polymerase holoenzyme right we already kind of

talked about that with the core enzyme two alpha beta beta prime omega and then the

the sigma subunit all of that's needed to bind to the promoter region and eukaryotic tells us a little bit

more right we said that we needed two things we needed an rna polymerase and which what are we making here

initiation and we're going to say that we're trying to make what we're trying to make mrna transcription

so we're doing transcription but we're making mrna so what was the particular rna polymerase

1 2 3 r m m is for r for the mrna so rna polymerase type 2 is one of the things that i need

the second thing that i need is the general transcription factors and there's just so many of these that i

don't know how important and how specific we really need to go into these i'll give you some of them but i just

want you to know that there's so many different types of them the main one if you had to remember

one specific out of the tons of them i want you to remember transcription factor

2 d this is the one that i really want you to remember and the reason why is this contains a structure called the

tata binding protein so this transcription factor 2d has a particular protein portion

which binds to the promoter region the tata box but there's many other reasons region

transcription factors and you can remember these by transcription factor 2 and there can be h there can be e

there can be f there can be a there can be b so there's tons of these dang things

so i don't know how important it really is to know that but the main one i want you to remember

is the transcription factor 2d all right so these are the things that we need in order for initiation to occur

so let's take for example we're going to have on one side eukaryotic cells will the eukaryotic

enzymes will bind and on this side the prokaryotic will bind right so let's say here we take for

example we'll make this prokaryotic rna polymerase we'll make this one blue

and we'll make the rna polymerase over here for the eukaryotic cells just for the heck of it

we'll make it orange okay just so we can distinguish the difference between these so what will happen this whole rna

polymerase holoenzyme will do what bind to the promoter what will allow it to bind what

subunit of it the sigma subunit and if you really wanted to go back you guys remember we made that pink

okay for the eukaryotic cells what do we need we need the rna polymerase type 2. we said we're going

to represent that with orange so here's going to be the rna polymerase

type 2 and then what else do we need we need those general transcription factors there's a

bunch of them but what's the particular one that i really want you to remember here

transcription factor 2 d which contains the tata binding protein so it binds to the tata box which is the

promoter region in the eukaryotic cells then allows the rna polymerase 2 to bind to the dna

now once the rna polymerase is bound to the dna it's going to start moving down the dna strands reading it

and making rna so we've now started the process of transcription that's all that's

happening here the next step is that once we've bound had

this rna so let's write these down here for the prokaryotic cell this would be the

we'll put rna polymerase and we'll put h for the holoenzyme and for that one up here this is going to be

rna polymerase type 2 right once this is bound and it's in the dna it's going to start

reading the dna as it reads the dna it'll make mrna that process by which it does that is called

elongation so let's write that down now so the next step is elongation to make

the mrna now within elongation a couple different things happens and this is thankfully the same in

prokaryotic cells and eukaryotic cells so thank the lord for that right so let's just say that we take

either one of these rna polymerases let's just for the heck of it we'll say here's your rna polymerase ii okay

here's your rna polymerase two and it's reading the dna the dna we already know has two strands

we're going to call this top strand here this top strand sonogram this top strand up here we're going to call this the

template strand so the template strand also sometimes referred to as the anti-sense

strand this strand down here we're going to call the coding strand

now when rna polymerases read dna the strand that they read is the template strand or the antisense strand

so that's the first thing i really need you guys to remember is that the rna polymerases

what strand do they read they read we're going to put the template strand or also referred to as what else

the antisense strand and that's the strand that they use to make the mrna they do not use the coding strand

so let's kind of put a little asterisk here that this is the strand that we're gonna read

now when it reads it it does it in a way that you guys if you guys watch our dna replication video this should be

so darn easy let's say here this end of the dna is the three prime end that means that this end is

the five prime end and remember one strand of dna

on this side should have a complementary anti-parallel strand on the other side which means that this is the three and

on here this has to be the five end on this side and this has to be the

three end on that side what happens is this rna polymerase when it binds into the dna

it does something very interesting it binds to the dna through the initiation process

and then opens up the dna who opened up the dna before it was that whole in replication it was that whole like

replication complex rna polymerase does that so the first thing we need to know is that rna

polymerase does what it opens the dna now in replication what else happened

you opened the dna and you had those single stranded binding proteins which keep it stable and kept it open right

rna polymerase does that on its own so it also stabilizes the single

stranded dna molecules right so it stabilizes the single strands then what was the enzyme in replication

that opened up to unwound the dna helicase rna polymerase has its intrinsic helicase activity so it also

unwinds the dna after it unwinds the dna then it starts reading the dna

so let's say here as it reads the dna in this direction three to five it'll make mrna

that'll be going in the opposite direction so it's going to read this 3 all the way to the 5 direction and as it

does that it starts synthesizing mrna right and this mrna will be synthesized in what direction

what will this be this starting point the five end and what would be this point

the three end so we know the next thing that the rna polymerase does whether it be in prokaryotic cells or

eukaryotic cells is it reads the dna from three to five

then it synthesizes rna from what direction guys five to three very

very important the last thing that you guys should be asking is okay zach you also

said that in replication the dna polymerases read the dna and then if there was an accident or a

mistake they would proofread it and then cut out the nucleotide what about rna polymerases do they do

that as well because it looks like they've done everything that was similar in dna replication

that's the one thing that's controversial so the only thing that's kind of relatively controversial

is is there a proof reading function we don't really know it's still subject to study

so that's one thing to remember if you want to compare this the proofreading function is somewhat

uncertain at this point in time all right so we have an idea now we've

read this dna and we've made rna i know we talked about this a lot in dna replication we're talking about it here

and sometimes it really can be confusing when you're saying five end three and i don't i don't i

don't freaking get what you're talking about zach so i want to take a quick little second

and explain what the heck i mean when i say it reads it from three to five and synthesizes it from five to

three a diagram i really think will clear this up for you let's take a second to understand what i

mean by reading the dna three to five and then synthesizing it five to three i think

it's really important to understand that so let's say here we have this strand of dna so this is

this is going to be our dna template if you will okay so this is our dna template on this

side the blue one and then this is going to be the rna that we're going to synthesize utilizing

the rna polymerase type 2 and eukaryotes are the rna polymerase hollow enzyme and prokaryotes

now when we're making this rna we have to read the dna in what direction the three end

to the five and what is the three and you guys remember the video on dna structure

it's the oh so this is going to be the three end what's the five end it's the phosphate

group so the phosphate group is going to be the five and so i have to read this

starting at the o h portion towards the five end where the phosphate is so the rna polymerase let's pretend i'm the rna

polymerase i'm walking right to do i find the three prime and i'm like oh there it is okay i'm gonna move

up oh i found the three prime five prime let me just fill this up oh i feel my nitrogenous base

the nitrogenous base that it feels is adenine so it picks into its little satchel of nucleotides it's like okay

this is adenine the complementary base for is thymine uh oh no that's not correct because you

guys know that if we're taking dna making rna what's the one nucleotide that

switches from dna and rna adenine is no longer complementary to thymine in the rna it is uracil

so the dna the rna polymerase will come read find the three end read the nucleotide and say oop okay

this is an adenine reach into its satchel of a bunch of nucleotides and pull out

uracil when it pulls that out it then puts the nucleotide in a particular orientation

what's the orientation we said it reads it from three to five and synthesizes it from five to three

what's the five end here's the nucleotide the five end is this

phosphate group the three end is this oh group so it's going to kind of flip the nucleotide the opposite

direction and make sure that the nitrogenous base here is what

uracil then when it does that it's going to go to the next one so it's going to continue it's going to go to the next

point here's where the next oh group would be right the three prime end

reads it finds that finds the nucleotide it says oh the nitrogenous base here is t let me reach into my satchel of a

bunch of different uh good old nucleotides i'm going to read it t goes with a

i'm going to put my nucleotide and i'm going to flip it where it's five prime end

going down three prime end pointing up and then the nitrogenous base which is complementary to the t

is a when it does that it then fuses the three prime end and the five prime end together making a

bond what is that bond called the phosphodiester bond and the same process occurs

so then it'll do what let's fix this three prime in there it'll then go go to the next nucleotide

here's the three prime end where the oh group is reads it finds the nucleotide says that

it's a g reaches into its satchel pulls out a nucleotide with the cytosine when it does it it

flips it to where the five end is on this side there's my phosphate the three prime end is upwards

and it says oh the nucleotide that goes with this is with the nitrogenous base c then it

says oh i have my phi prime n situated close to the three prime end of the preceding

nucleotide let me fuse these two together and make my phosphate ester bond

and just for the heck of it because repet repetition i guess is helpful we go reads this says okay next one

here's my three prime end where the oh group is read it find the nitrogenous base it's a

cytosine digs into its satchel pulls out the nucleotide guanosine

sorry the guanine nitrogenous base then when it does that it situates it where the five prime end

is situated down three prime n is situated upwards in this case and then

the nitrogenous bases on guanine then it says oh my five prime n i can stitch it to the three prime end of the

preceding nucleotide and form my phosphodiester bond and that's how we make rna

reading it three to five and synthesizing it from five to three dang we good all right

now that we've done that the last thing i need you to understand is that rna polymerase is a very important

enzyme within eukaryotic and prokaryotic cells a question that can come up and it's so

dumb and annoying but you should know it is that in eukaryotic cells we can inhibit the rna polymerase

by using a kind of toxin amanitin it's for mushrooms and this can inhibit the rna polymerase

within we'll put eukaryotic cells okay there's another drug which they love to ask in the exams as

well called rifampicin it's an antibiotic and this inhibits the rna polymerase

within if it's an antibiotic that's good against bacteria prokaryotic cells so this will inhibit the rna polymerase

within prokaryotic cells which would inhibit what the part of the initiation

the elongation basically making rna if you can't make rna you can't make proteins

if you can't make proteins you can't perform the general functions of the cell

so this is kind of from a poisonous mushroom which is stupid to know that but they like to ask it on your exams

and then rifampicin is an antibiotic which they also love to ask okay now we've talked about elongation

we've made the dang rna rna polymerase is working real hard the last thing we got to do is we got to

just end it we don't need any more rna we've made the rna that we need to make the

protein that is called termination all right so we talked about elongation the next step

the last step really that we got to discuss here is termination we've got to end this whole

transcription process so the last step is termination now unfortunately

termination is probably one of the more annoying and complicated ones unfortunately

and it is different in prokaryotes and eukaryotes that's why it kind of makes it a little bit frustrating

but termination is basically where we've already made our rna transcript and we just need to

detach it or disassociate it away from the dna and prevent that rna polymerase from reading

any more of the dna and making any more rna so just stop transcription how do we do that in

prokaryotes there's two mechanisms one of the ways that this happens is through what's called

road dependent termination so one is via this process called row dependent termination and it's really

simple believe it or not so let's say here we take the prokaryotics we we picked blue for our

rna polymerase so the rna polymerase here's our rna polymerase it's reading this dna as it's reading

the dna again what is it making from it you guys remember it's making the rna in this case

it could be any rna it could be the mrna trna rna whatever as it does this there's a protein called

rho and what rho does is this rho protein will start moving up the mrna

and as it moves up the rna that's being synthesized by the rna polymerase as it gets to this rna polymerase it basically

says hey it just punches the rna polymerase off the dna

if you punch the rna polymerase off the dna is it going to be able to continue to

keep breeding the dna and making any more rna no so that terminates the transcription process

so the big thing i need you guys to know here is that with the road dependent termination

is rho protein causes rna polymerase uh to break away to disassociate if you

will okay to break away from the dna okay

all right beautiful the next mechanism within prokaryotes is rho independent termination so we

don't use the row protein so we call this row independent termination now with this

process it's a little bit more complicated and a little annoying let's say here we have the dna right and

within the dna we're going to mark these here we're going to say this is our template strand right so this strand is

the template strand right or the antisense strand and then this is going to be our coding strand

so which one does the rna polymerase read it reads the template strand or the antisense strand

there's a particular like thing called inverted repeats that form within the dna that the rna polymerase is reading

so what happens is this rna polymerase will bind to that template strand and it'll start

reading it making the rna as it starts making this rna

it it encounters a particular sequence of of dna called inverted repeats let's

write these inverted repeats out in kind of a nice little color let's do let's do orange

and let's say here we have an inverted repeat where we have c c g g and then a bunch of nucleotides

that we don't care about and then here we'll have ggcc okay then we're just going to have this

is the template again on the coding strain it would just be the complementary base so if this was cc

this would be gg cc we don't really care about these nucleotides cc

gg right the rna polymerase is going to read this template strand what happens is right you're going to get this kind

of strand here where you'll have a bunch of nucleotides already kind of made up here

and then it reaches this kind of like inverted repeat area and what happens is it reads this and

then basically everything you read within the template strand should be the same as it is in the coding strand

because it's the complementary base so you'll have g g c c that it'll make a bunch of nucleotides

we don't care about and then c c g g what happens is whenever this

rna is kind of coming and being transcribed from the rna polymerase something interesting happens where some

of these c's and some of these g's on this portion have a strong affinity for some of the c's and some of the g's

in this portion of the rna and as they start having this affinity they start approaching and kind of wanting to

interact with one another via these hydrogen bonds and so it creates this really

interesting kind of like hairpin loop if you will where there's a bunch of g's and c's

within this kind of hairpin loop that are kind of interacting with one another and what happens

is that hairpin loop is what triggers the rna polymerase to pretty much

hop off of the dna and terminate the transcription process because what happens is once you form

this hairpin loop what will happen is there's going to be particular enzymes that will

bind to that portion and cleave the d the rna away from the rna polymerase so the big

thing i need you to know within row independent termination is that you'll hit this area the rna

polymerase will be transcribing reading the dna making rna it'll hit these areas of inverted repeats

when these inverted repeats are made they create this thing called a hairpin loop this hairpin loop

will then trigger particular cleavage enzymes to come and cleave a couple nucleotides

after that hairpin loop to cleave that away from the rna polymerase

and then here you have your rna that you formed so that is one of the ways that we have

termination road independent via prokaryotes the last termination mechanism is going to be

eukaryotic cells now how does this work this one's actually relatively simple so we had the rna polymerase in eukaryotes

and this was orange okay it's binding to the dna it's reading the dna

as it's reading the dna it's making rna as it starts making this rna it hits a particular sequence

where when it starts reading the dna and makes rna it makes a particular sequence of

a a u a a a okay so what are the what is the nucleotide sequence here let's write it

out this portion here will be double a u triple a this is what's called a

polyadenylation signal so what is this called here this is called a poly

adenylation signal and once this kind of nucleotide sequence occurs

so it's kind of now that we know what that nucleotide sequence is let's kind of just put like this

here's that nucleotide sequence that polyadenylation signal that's been synthesized or formed by the

rna polymerase with the eukaryotes once that happens it activates particular

enzymes and those enzymes will come to the area here and cleave the rna away from the rna

polymerase separating out this rna away from the dna and the rna polymerase and

then again what will i have at this portion here just as kind of a diagrammatic portion

here this will be my polyadenylation signal this is important because we're going to talk about

post-transcriptional modification in a second so i know this was a lot of crap just

really quickly recap because this is one of the toughest parts of transcription

is termination prokaryotes there's two ways road dependent row independent with this one you need a row protein to

knock the rna polymerase off if you don't have him he can't make any more rna

the other one is row independent you don't have a row protein the rna polymerase is reading the dna

making rna and it hits these areas of inverted repeats these inverted repeats when they're made within the

rna it creates a hydrogen bond interaction between them which causes it to loop forming a

hairpin loop that signals particular enzymes to break the rna

away from the rna polymerase and we've made our rna there the last one is in eukaryotes the rna

polymerase is reading the dna and it reaches a particular sequence of nucleotides where it reads

and then makes a a u triple a a polyadenylation signal which activates enzymes to come

cleave the rna away from the rna polymerase terminating the transcription process that really

hammers this home let's now talk about post-transcriptional modification we know at this point how to take dna

make rna right we talked about all the different types of rna utilizing rna polymerases

utilizing the transcription factors we talked a little about a gene regulation we even went through all the stages of

transcription taking the dna and making the mrna all the way up until the point where we

finally made the mrna and broken it away from the dna unfortunately that's not it for

transcription now we have this mrna right so we basically what have we covered up to

this point we took the dna we read let's just say here

at this portion i'll just put here's our promoter our rna polymerase has read this gene

sequence we hit a termination sequence let's say here's our termination sequence

that we talked about here and once we hit that termination sequence the rna polymerase will fall off and

then from this you'll make the rna so this was pretty much the basic aspects of the transcription

but now we got to modify this now here's the thing it's actually kind of a misnomer to say

that this is mrna it's technically not mrna right now

so this piece of rna that we made okay and this is this process of post-transcriptional modification

this only occurs it's very important let me actually write this down this only occurs in

eukaryotic cells so that's nice all this stuff that we're going to talk about here is only in eukaryotic cells

it doesn't happen in prokaryotic cells so they just make their rna and that's it so technically

this immature mrna if you will we actually give it a very specific name we call it heterogeneous

nuclear rna now this heterogeneous nuclear rna is kind of an immature

mrna that has to go through some modifications to really make mature mrna that then can be translated to make

proteins what are those modifications the first thing that we have to do

is we have to put something on one of these ends so now we got to know a little bit about the

terminology of the ends of this immature mrna or hn rna on this end we're going to call this the five prime

end what's on that five prime end do you guys remember the phosphate groups what's on this end

the three prime end what's on the three prime mint the oh group okay now

something very interesting is on the five prime end on the five prime end of this heterogeneous nuclear rna or the

hrna you have a triphosphate which we're representing here with these

orange circles an enzyme comes to the rescue and cleaves off one of those phosphate molecules what is

that enzyme called it's this orange little cute enzyme this orange enzyme is called

rna tri-phosphatase and what it does is it comes and cleaves off

what portion it cleaves off one of these phosphate groups it's going to cleave off one of the phosphate groups

so now i only have two phosphates on the end of this five prime end then another

enzyme comes in and it says hey there's only two phosphates here

i can now add something on here and i'm going to add on what's called a gmp molecule what am i going to add on

again i'm going to add a gmp molecule which is guanosine monophosphate so we're going

to represent that here which we add on the phosphate for the guanosine monophosphate

and then we're going to just represent this as the guanosine so this is our guanosine and that blue circle there is

the phosphate on the guanosine so what does he add on technically he adds on to this little two phosphates

right it adds in gtp but when it does that two phosphates are

released in the form of pyrophosphate which then get broken down by pyrophosphatase into

individual phosphates so if i took gtp and i removed two phosphates what am i left with

gmp so it adds on this gmp group onto that two phosphate end on the five prime end

so this enzyme that adds that gmp on in the form of gtp is called guanolile

uh transferase guanalyl transferase beautiful so this last enzyme here which is

involved in this step here on the five prime n is going to add on a methyl group onto

one of the components of the guanosine monophosphate it's actually like one of

the seventh components on that structure it adds on a methyl group

and so at the end of this this enzyme which adds a methyl group on what do you think it's called methyltransferase

at the end of this process where you took the prime end which had three phosphates got rid of one

took the guanola transfers added on the gmp took the methyl transferase added on that methyl group

you formed this complex here and we call this whole complex that we just added on a seven methyl guanosine group

okay and that's on that five prime end this is called capping this is called capping so whatever we've

just done on this five prime end is called capping what the heck do we do all this stuff for

the whole purpose of capping is to help to initiate translation so this sequence this kind

of five prime end with that seven methyl guanosine or that five prime capping if you will

it's kind of a signal sequence if you will that allow for it to interact with the ribosome

and undergo translation the other thing it does is it prevents degradation by

nuclease enzymes that want to come and break down the rna so it helps to prevent

degradation helps to initiate the translation process one more thing that they it's a dumb

thing to know but they love to ask it is that there is a particular molecule that this methyltransferase

uses to add that methyl group on and sometimes it's really important to know it

and this is called s adenosyl methionine also known as sam sam carries a methyl group it's like

a methyl donor if you will it gives that methyl group to the methyl transferase

and the methyl transferase adds that methyl group onto the guanosine monophosphate forming the

7-methylguanosine or that 5-prime cap okay so that's the first thing that happens

now we got to talk about the 3-prime end on the three prime end we had that oh group right that's the ohn but do you

remember in eukaryote there was a particular signal

that prevent that generated that terminated transcription what was that nucleotide signal do you guys remember

test your knowledge guys a a u triple a right that was that

polyadenylation signal do you guys remember that the polyadenylation segment we talked about in eukaryotes