RNA Transcription 3b'

Summary

This lecture delves into the intricacies of transcription and RNA processing, focusing on the initiation phase and the pivotal role of sigma factors in recognizing promoter sequences. It discusses the transition from initiation to elongation, akin to DNA replication, where RNA polymerase reads the template strand and synthesizes RNA. The session also covers the mechanisms of transcription termination in prokaryotes, contrasting intrinsic and rRNA-dependent termination. The lecture further explores the complexities of eukaryotic transcription, highlighting the involvement of multiple RNA polymerases, general transcription factors, and the role of enhancers and silencers in regulating the process.

Takeaways

• 🔬 The lecture discusses the process of transcription and RNA processing, focusing on the initiation, elongation, and termination of transcription in both prokaryotes and eukaryotes.
• 🌟 The importance of the sigma factor in prokaryotes is highlighted, as it recognizes promoter sequences and initiates transcription.
• 🧬 Elongation in transcription is compared to DNA replication, with RNA polymerase moving along the template strand and synthesizing RNA in a 5' to 3' direction.
• 🔄 Termination of transcription in prokaryotes can occur through two mechanisms: intrinsic (row-independent) and extrinsic (row-dependent), involving specific sequences and proteins.
• 🧲 Intrinsic termination involves a hairpin loop and a string of A's on the template strand, which causes RNA polymerase to pause and release the RNA.
• 🔄 Extrinsic termination in prokaryotes relies on the rho protein, which binds to the RNA at a specific site and disrupts the DNA-RNA hybrid to terminate transcription.
• 🌐 Eukaryotic transcription is more complex, involving a larger genome, the presence of a nucleus, and the need for RNA processing and export to the cytoplasm.
• 🧬 Eukaryotes have three different RNA polymerases, each with distinct promoter sequences and functions, including the transcription of ribosomal genes, small RNA genes, and protein-coding genes.
• 🔑 Transcription factors play a crucial role in eukaryotic transcription, binding to promoter elements and facilitating the binding of RNA polymerase to initiate transcription.
• 🔋 The process of transcription initiation in eukaryotes involves a specific order of transcription factor binding, leading to the formation of a pre-initiation complex and the start of transcription.

Q & A

What is the role of the sigma factor in the initiation of transcription?

The sigma factor is crucial for the initiation of transcription as it helps the RNA polymerase recognize and bind to the promoter sequences, specifically the -10 (Pribnow box) and -35 regions, enabling the start of RNA synthesis.

How does the elongation process of transcription differ between DNA replication and RNA synthesis?

Although elongation in transcription is similar to DNA replication, the key difference is that RNA polymerase moves along the DNA template strand from 3' to 5', synthesizing RNA in the 5' to 3' direction using ribonucleotide triphosphates instead of deoxyribonucleotides.

What is the function of the 'transcription bubble' mentioned in the script?

The 'transcription bubble' is a region of local denaturation that forms behind the RNA polymerase as it moves along the DNA template. It consists of the single-stranded DNA and the newly synthesized RNA, and it allows for the continuous synthesis of RNA while the DNA re-anneals behind the bubble.

What are the two mechanisms of transcription termination in prokaryotes?

The two mechanisms of transcription termination in prokaryotes are Rho-independent termination and Rho-dependent termination. Rho-independent termination involves the formation of a hairpin loop and a string of U's in the RNA, causing the RNA polymerase to pause and release the RNA. Rho-dependent termination requires the Rho protein, which binds to a specific site on the RNA and moves along it until it reaches the RNA polymerase, causing termination.

What is the significance of the poly U stretch in RNA during Rho-independent termination?

The poly U stretch in RNA, resulting from a string of A's on the template strand, plays a role in Rho-independent termination by creating weak A-U base pairing that facilitates the release of the RNA from the DNA template, thus ending transcription.

How does the Rho protein contribute to Rho-dependent termination of transcription?

The Rho protein contributes to Rho-dependent termination by binding to the RNA at the Rho Utilization Site (RUT site), using ATP to move along the RNA, and catching up with the RNA polymerase. It then sterically hinders or unwinds the DNA-RNA hybrid, causing the release of the RNA transcript and termination of transcription.

What are the differences between prokaryotic and eukaryotic transcription in terms of complexity?

Eukaryotic transcription is more complex than prokaryotic transcription due to the presence of a larger genome with more genes and non-coding DNA, the necessity of RNA export from the nucleus to the cytoplasm, and the presence of chromatin. Additionally, eukaryotes have three different RNA polymerases, each with their own set of genes to transcribe, and require processing of precursor mRNA, including the removal of introns and addition of exons.

What is the role of the TATA box in eukaryotic transcription?

The TATA box, also known as the Hogness box, is a DNA sequence element found in the promoter region of many eukaryotic genes. It plays a crucial role in the initiation of transcription by providing a binding site for the TATA-binding protein (TBP) and other transcription factors, which help assemble the transcription pre-initiation complex.

How do enhancers and silencers regulate transcription in eukaryotes?

Enhancers and silencers are regulatory DNA sequences that can be located at a distance from the promoter and can influence the rate of transcription. Enhancers bind activator proteins that stimulate transcription, while silencers bind repressors that inhibit transcription. They can modulate the transcription machinery's assembly and function, either promoting or suppressing gene expression.

What is the significance of the order in which transcription factors bind during eukaryotic transcription initiation?

The order in which transcription factors bind is significant as it ensures the proper assembly of the transcription pre-initiation complex. For example, transcription Factor 2D (containing TBP and TAFs) binds the TATA box first, followed by the binding of other factors like 2B, RNA polymerase II, and then 2F and 2H, which together form the complete initiation complex and trigger the start of transcription.

Outlines

00:00

🔬 Transcription and RNA Processing

This paragraph discusses the process of transcription initiation, emphasizing the role of the sigma factor in recognizing promoter sequences (Theus 10 and Theus 35) and binding to the core RNA polymerase. It explains how the sigma factor can be released once transcription starts, transitioning into the elongation phase. Elongation is compared to DNA replication, with RNA polymerase moving along the template strand, synthesizing RNA using ribonucleotide triphosphates. The paragraph also describes the formation of a transcription bubble, where the newly synthesized RNA and the DNA separate as transcription proceeds. Finally, it introduces the concepts of intrinsic and Rho-dependent termination mechanisms in prokaryotic transcription, detailing how RNA polymerase recognizes termination sequences to end transcription.

05:03

🧬 Intrinsic and Rho-Dependent Transcription Termination

This section delves into the specifics of intrinsic (Rho-independent) and Rho-dependent termination in prokaryotes. Intrinsic termination involves the formation of a hairpin loop and a string of Us in the RNA, which cause the RNA polymerase to pause and eventually release the RNA, terminating transcription. Rho-dependent termination, on the other hand, requires the Rho protein binding to a specific site in the RNA, which then moves along the RNA and interacts with the RNA polymerase to terminate transcription. The paragraph illustrates these mechanisms with figures and explains how mutations in the sequences involved can affect the termination process.

10:04

🌐 Complexity in Eukaryotic Transcription

The paragraph contrasts eukaryotic and prokaryotic transcription, highlighting the increased complexity in eukaryotes due to a larger genome, the presence of a nucleus, and the need for RNA processing and export. It mentions that eukaryotes have three different RNA polymerases, each responsible for transcribing specific types of genes. The paragraph also discusses the structure of eukaryotic promoters, which can vary and may include elements like the TATA box, CAT box, and GC-rich regions. These elements are recognized by general transcription factors that facilitate the binding of RNA polymerase and the initiation of transcription.

15:05

🧠 Eukaryotic Transcription Initiation and Regulatory Elements

This part focuses on the initiation of transcription in eukaryotes, detailing the order in which transcription factors bind to the promoter region. It describes the role of the TATA box and other promoter elements in facilitating the binding of RNA polymerase and the assembly of the transcription machinery. The paragraph also introduces the concept of enhancers and silencers, which are regulatory sequences that can modulate the transcription process by binding activator or repressor proteins, respectively. The discussion includes the role of the carboxy-terminal domain of RNA polymerase in the initiation of transcription and how phosphorylation of this domain can trigger the start of transcription.

20:06

📚 Elongation and Regulation of Transcription

The final paragraph discusses the elongation phase of transcription in eukaryotes, which is similar to prokaryotes, with RNA polymerase reading the template strand and synthesizing RNA. It also touches upon the role of enhancers and activator proteins in regulating transcription, providing an example of the yeast UAS (Upstream Activator Sequence) as a strong enhancer. The paragraph briefly mentions that the next lecture will cover termination of transcription, suggesting a continuation of the discussion on the regulation of gene expression.

Keywords

💡Transcription

Transcription is the process by which the genetic information from DNA is copied into RNA, specifically messenger RNA (mRNA), in preparation for protein synthesis. In the video, transcription is a central theme, with detailed discussions on how it is initiated, elongated, and terminated in both prokaryotic and eukaryotic cells. The script describes the role of RNA polymerase and the significance of promoter sequences in starting transcription.

💡Sigma Factor

The sigma factor is a protein that associates with RNA polymerase and is crucial for the initiation of transcription in prokaryotes. It helps the RNA polymerase recognize and bind to specific promoter sequences on DNA. The script mentions the importance of the sigma factor in recognizing the -10 (Pribnow box) and -35 regions of the promoter, which are essential for the start of transcription.

💡Elongation

Elongation refers to the phase of transcription where RNA polymerase moves along the DNA template, synthesizing the RNA molecule by adding complementary ribonucleotides. The script likens this process to DNA replication, highlighting the polymerase's movement in a 3' to 5' direction along the DNA and the 5' to 3' synthesis of the RNA strand. Elongation is a critical step as it determines the length and sequence of the RNA transcript.

💡Termination

Termination is the end of the transcription process, where RNA synthesis ceases. The script explains two mechanisms of termination in prokaryotes: Rho-independent termination, which involves the formation of a hairpin loop and a string of Us in the RNA, and Rho-dependent termination, which requires the Rho protein to bind to an RNA sequence and facilitate termination. Understanding termination is key to knowing how genes are regulated at the transcriptional level.

💡Promoter

A promoter is a DNA sequence that signals the start of transcription. The script discusses how promoters contain specific sequences like the -10 and -35 regions in prokaryotes and the TATA box in eukaryotes, which are crucial for RNA polymerase binding and the initiation of transcription. Promoters are essential for gene regulation, determining which genes are transcribed and when.

💡RNA Polymerase

RNA polymerase is the enzyme responsible for catalyzing the synthesis of RNA from a DNA template during transcription. The script describes its role in both prokaryotic and eukaryotic transcription, highlighting its movement along the DNA template and the synthesis of the RNA strand. RNA polymerase is a key player in gene expression, as it determines which genes are transcribed into RNA.

💡Eukaryotic Transcription

Eukaryotic transcription refers to the process of RNA synthesis in eukaryotic cells, which have a more complex system compared to prokaryotes. The script contrasts eukaryotic transcription with prokaryotic, noting the presence of a nucleus, the need for RNA processing (such as splicing), and the involvement of three different RNA polymerases, each with specific roles. This process is intricate and involves multiple steps beyond the basic transcription mechanism.

💡Introns and Exons

Introns and exons are terms used to describe parts of eukaryotic genes. Introns are non-coding sequences within a gene that are removed during the processing of pre-mRNA, while exons are the coding sequences that are retained and eventually make up the mature mRNA. The script mentions that eukaryotic genes contain both introns and exons, and that processing of the precursor mRNA, including the removal of introns, is necessary before it can be exported to the cytoplasm for translation.

💡General Transcription Factors

General transcription factors are proteins that assist the RNA polymerase in the initiation of transcription in eukaryotes. The script explains that these factors, labeled A through H, interact with RNA polymerase II to help it bind to the promoter and initiate transcription. Unlike prokaryotes, which use a sigma factor, eukaryotes rely on multiple transcription factors to facilitate the binding and initiation process.

💡Enhancers and Silencers

Enhancers and silencers are regulatory elements in eukaryotic genomes that modulate the transcription of genes. Enhancers, as mentioned in the script, are sequences that bind activator proteins and increase the rate of transcription, while silencers bind repressor proteins that decrease transcription. These elements can be located far from the promoter and play a crucial role in the regulation of gene expression, allowing for precise control of when and where genes are transcribed.

Highlights

Initiation of transcription is crucial, and the sigma factor plays a key role in recognizing promoter sequences.

The core RNA polymerase and sigma factor bind to the promoter region, initiating transcription at specific sites.

Elongation of RNA is similar to DNA replication, with RNA polymerase moving along the template strand and adding complementary nucleotides.

Termination of transcription in prokaryotes can occur through intrinsic or Rho-dependent mechanisms, involving specific sequence recognition.

Intrinsic termination involves a hairpin loop and a string of Us in the RNA, causing the RNA polymerase to pause and release the RNA.

Rho-dependent termination requires the Rho protein, which binds to the RNA and moves along it, eventually disrupting the DNA-RNA hybrid.

Eukaryotic transcription is more complex due to a larger genome, the presence of a nucleus, and the need for RNA processing and export.

Eukaryotes have three different RNA polymerases, each responsible for transcribing different sets of genes.

Promoters in eukaryotes have multiple recognition sites, including the TATA box, CAT box, and GC-rich boxes, which interact with general transcription factors.

The order of transcription factor binding in eukaryotes is crucial for the initiation of transcription.

Transcription Factor 2D, containing the TATA binding protein, is the first to bind, initiating the formation of the pre-initiation complex.

The carboxy-terminal domain of RNA polymerase 2 is phosphorylated, triggering the start of transcription.

Enhancers and silencers are regulatory sequences that can modulate transcription by binding activator or repressor proteins.

The yeast UAS is an example of a strong enhancer that can significantly increase transcription when present.

Elongation in eukaryotes follows a similar process to prokaryotes, with RNA polymerase synthesizing RNA along the DNA template.

Transcripts

all right this is the second part of the

transcription and RNA processing

recorded lectures so we were talking

about initiation of transcription

importance of the sigma factor and being

able to

recognize the promoter sequences Theus

10 and Theus

35 so here we have the core RNA plase

Sigma factor in there and recognizing

the promoter region here Theus 35 Theus

10 to get to the RNA plas where we can

start transcribing

RNA I talked about how when um that

starts the sigma Factor can be released

because its job is done we then move on

to elongation and elongation is very

similar

to DNA replication we can look just take

quick look at this slide here so you

have the template Strand and again three

prime here five Prime here so RNA

polymerase is moving

down a template strand it's reading it

so to speak in a 32 five Prime Direction

okay going down this way and then it's

bringing in nucleotides that are

complimentary to the template Strand

and again it uses ribonucleotide

triphosphates it'll cleave off two those

phosphate groups pyrro phosphate so that

you can form the energy to form that

that calent Bond

there so this just shows again you got

the coating strand or the non-template

Strand has the uh same sequence as RNA

made u c a u or t c a and going along

the template strand there so RNA plary

slides along the

DNA goes to the template strand making

complimentary RNA um slides along 3 to

five Prime Direction so it can

synthesize the RNA in 5 to3 Prime pyrro

phosphate is lease we just looked at

that in the last slide and same um base

pairing rules except that you have U

instead of T um so again um elongation

occurs very much like DNA

replication it moves along um the DNA

strand three prime to five prime it

moves about in procaryotes about 30 to

50 nucleotides per second um you give

you an idea DNA plase moves about 2,000

nucleotides per second so DNA

replication much faster than

transcription as the pmer moves along it

creates what I've mentioned before a

transcription bubble um behind the

bubble the DNA DNA reenal and displaces

the newly synthesized RNA so as the DNA

starts to reenal here the RNA the red

Parts displaced outside of the

bubble all right so let's get

to then termination of

transcription so how does RNA plase know

when it's reached the end of a gene when

to stop transcription well it knows when

it reaches a termination

sequence and there are two mechanisms by

which this can occur in procaryotes

there is the intrinsic or row

independent

termination and there is then the second

way the row dependent

termination so we'll talk first about

the intrinsic or row independent

termination so the Terminator sequence

in R independent termination consists of

a string of A's on the template strand

resulting in a string of U's or a poly U

stretch in the RNA that is

made this um termination sequence also

consists of a sequence of two-fold

symmetry which allows a hair pin form

Upstream of the A's and use okay

Upstream again to the left or to the

five Prime end the sequence of twofold

symmetry is G and C rich and forms a

complimentary intramolecular hydrogen

bonds taking on the form of a hairpin

Loop that's followed then by this run of

use in the RNA now remember RNA pulas is

about 50 nucleotides long RNA plase will

move down the DNA this hair pin is

thought to slow down the plase somewhat

as it's going along and causing the

plase to pause and when the plase pauses

this weak a and u DNA RNA base pairing

releases the RNA thus ending

transcription so again words sounds kind

of funny let's take a look at how this

is happening here so here's the

DNA here's RNA plas moving down at some

point it transcribed over here this GC

rich sequence that had this um com that

can that had this two-fold symmetry in

it so it could form complimentary

intermolecular hydrogen bonds and so it

forms this hair pin Loop all right and

as RNA polymerase is going it makes

hairpin Loop RNA polymerase it's this

hairpin Loop sort of get stuck in the

RNA plates it slows down and then it

makes all these U's and A's or the A's

and the DNA U's and the RNA here and you

have this weak unaa binding and it falls

apart and so termination happens so

here's

another picture here sort of showing it

so here's these again row independent

termination G's and C's Rich um

complimentary sequences so as

transcription takes place transcribe

this region transcribe that region it

folds up as a hair pin Loop and again

RNA polymerase is huge it's way like

this here and this hair pin Loop gets

stuck in RNA plas and so it transcribes

this 's and a as it gets stuck it pauses

these dissociate transcription then is

terminated here's another figure here

showing this um hairpin Loop so here's

the twofold Symmetry here there's a

transcription they can form this hairpin

Loop because these G's and C's

complimentary base be and so the

question is how do scientists know this

really is necessary for termination well

they know because as you can see here

they made mutations in these G's and C's

region which didn't allow a hairpin Loop

to form and when they did that when they

mutated these so the herin loop conform

then termination didn't happen correctly

okay in the same way if they deleted a

bunch of the uh A's appear they used in

form and they also then didn't get

proper termination so they have a

hypothesis they look at the sequence and

say hey look what's happening maybe this

is responsible or you go and then you do

the experiment say yeah if we with this

it no longer works

right okay so that was an intrinsic or

row independent termination the second

kind of termination is row dependent

termination for genes that are

terminated by row dependent termination

they do not have that polya sequence so

they don't form a

polyu sequence in the RNA and they often

don't have a hair pin region sometimes

they do but often times they don't here

the row protein which is a hexamer so

it's six parts put together binds to the

RNA at the the rut site so the row

protein binds to the rut site the rut

site stands for row

utilization site okay R for row UT for

utilization site um so it's a site where

the row protein utilizes or binds to the

RNA so what ends up happening um is this

row or sorry this rut site is just

Upstream of the termination site while a

polymerase pauses again at the

termination site again if there's a

herin Loop or for some unknown other

reason the row protein binds to this rut

site and slides along the RNA it uses

ATP hydroly ATP for energy to slide

along the RNA it then catches up with

the RNA

plase and somehow sterically hinders it

to disrupt the DNA RNA hybrid which then

releases the transcript or stops

transcription so let's take a look at

what that looks like in some figures so

here's the DNA here there's the row

recognition site or utilization site

gets transcribed in the RNA PL keeps

going okay

somehow it pauses here this row protein

binds and it starts using ATP to move

along here it reaches and during the

pause it reaches the RNA here causing

that complex to separate I have another

figure showing very similar

thing is

transcribing here's the doesn't show the

rut site but the RO hexer binds here so

it goes pauses at the Terminator Okay

the p is pause Terminator the row

proteins moving up here unwinds that DNA

RNA hybrid somehow or sterically hinders

something which separates RNA from the

DNA then terminates okay so the exact

I'm not completely clear on the exact

mechanism um when I learned in grad

school was thought it was something sort

of steric hindrance that that row

protein had with the plase we've been

reading different places and in this

slide where it's um unwinding the DNA

RNA hybrid but it's doing something to

cause termination of transcription at

that point by separating the DNA from

the

RNA all right so let procaryotic

transcription again there's going to be

lots of similarities between procaryotes

and UK carots and let's cover some of

the differences here now uh eukariotic

transcription is more complicated than

procaryotic transcription just like UK

arotic replication was more complicated

than procaryotic replication UK carots

have a larger

genome consisting of more genes and more

non-coding DNA while eoli has 900 genes

per 1 million base pairs ukar only have

nine genes per million base pairs so a

lot of extra DNA UK carots also have a

nucleus all right therefore RNA must be

exported out into this cytoplasm somehow

so this isn't really transcription but

what happens after the RNA is

transcribed there's another set of

events that have to occur to get that

RNA out into the

cytoplasm in addition to the larger

genome genes have exons and introns that

must be processed in the nucleus before

RNA can be exported okay so another step

so it doesn't really influence the

transcription per se but there's a

processing step that has to occur to

remove parts of the RNA before the RNA

can be moved out into the cytoplasm UK

carots also have chromatin okay or they

have more protein wrapped around their

DNA so that DNA is wrapped around these

histone proteins and there's some

regulation of the transcriptional events

that is also that is actually due to

those proteins or the chromatin

structure so there just more

complexities into uh the control of

transcription then there are three

different RNA polymerases made up of

several polypeptides and they each

transcribe their own set of genes so

there's one RNA plase for procaryotes

there are three for ukar so RNA plase

one transcribes mainly ribosomal genes

or R RNA genes um we'll talk about R

rnas in ribosomes in a minute or in a

little bit here um but there's the 18s

5.8s and 28s ribosomal genes that are

transcribed by RNA plas 1 R plase 3

transcribes mainly small RNA genes um

they transcribe T rnas they transcribe

the 5sr RNA and they transcribe these SN

rnas are the small nuclear RN the plase

we're going to focus on um mainly is RNA

plase 2 okay middle one here because RNA

p 2 transcribes protein coding genes so

they transcribe

mrnas and a few uh small nuclear rnas as

well but primarily they are the the RNA

Pras 2 is the plase that transcribes

protein coding genes and will be the

main focus of what we talk about we'll

just give some mention RNA plase 1 and

three so each of these RNA plas is

recognized as their own promoter

sequence RNA plase 1's promoter is not

well characterized and it's uh but it is

Upstream of the start site while RNA

pmer 3 promoter is actually internal to

the gene so it's actually Downstream or

yes sorry it's Downstream of the start

site which is different than what we

were talking about RNA Pates to promot

will be upstream and what we'll talk

about here so just a couple of images

here um just threedimensional structures

of RNA plases you can see form this

clamp like structure the donut sort of

shape this opening here that surrounds

the DNA similar to DNA plases you see it

opening here and the DNA being red in

the the middle there so just a little

bit of idea of how it binds um couple of

differences then in uh procaryotic

versus eukariotic transcription it's RNA

plase makes RNA that RNA can then

immediately be used for translation in

bacterial cells there's no membrane

bound

nucleus and yeah whereas in UK carots

right you get this membrane bound

nucleus then you also have precursor

mRNA which we talk about you have

introns and exons so you got to get rid

of those introns we'll talk more about

this so there's these processing steps

and then you got to get that RNA out

into the cytoplasm where transcription

can then occur

all right so transcription of protein

coding genes or transcription by RNA

plase 2 so promoters for protein coding

genes are best characterized in UK carot

RNA plas 2 transcription plus one again

is the initiator region right the first

nucleotide put on and when transcription

starts the promoter region has several

different sites in it and all these

sites don't necessarily have to be there

some can be there some can't we'll talk

about the sort of the best or most

well-known sites um the first site on

the negative side there - 255 or about

-30 is the Tata box or hoges box it's

called the Tata box because that's the

consensus sequence ta ta pretty similar

to the prol box that has lots of T's and

A's in it slightly different location

hogness just the name of the individual

that um was responsible for discovering

that um sequence um further Upstream -

80 - 90 or a couple more what you call

proximal promoter elements proximal

means close to so close to the start

site the initiator region um and these

are called the cat box and the GC Rich

boxes um cat box just because that's a

consensus sequence there c a a and GC

Rich boxes are exactly that they have

lot of G's and C's um and that could be

variable you can have long regions

several GC boxes and again you might

have some or not yet

and um I forgot to point out this

initiated region is not only the

location where initiation occurs but

there's also uh sequences in there that

are thought to play a role in this start

of transcription so recognized um within

the

promoter um so there's again there's

this great variation in eukariotic

promoters um not all promoters have all

of these sequences so promoter sequences

in ukar are recognized by transcription

factors or what are called General

transcription factors to to facilitate

RNA plas to binding so where Sig where

the sigma factor helped RNA plas bind in

procaryotes in UK carots there's going

to be a multiple uh mult there going to

be multiple numbers of transcription

factors General transcription factors

and that are going to help RNA plas bind

to the promoter and start transcription

they are named um a through H so trans

they're labeled here transcription

Factor 2A means general transcription

factor that interacts with um RNA plase

two so transcription Factor two

interacts AR p 2 a you have

transcription Factor 2 b c d and so on

um so if they're transcription factors

that interacted with RNA plas 1 they'd

be transcription Factor 1 a and so on

and so forth so the the number here say

tells you which um RNA plase they uh

interact with so here we go um start

side transcription plus one so there's a

region here that helps within the

promoter minus 25 you got the Tata box

there further Upstream then GC boxes Cat

boxes can be present all of which are

helping with um The Binding of RNA plase

so that transcription can start um so

initiator region position Tata box C box

GC box relative positions where they can

be found um transcription factors that

bind to them we'll talk about uh Tata

binding protein here this um One that

binds to the initiated region in the

Tata box mainly there are also other

various transcription factors that are

involved in binding to these some of

these other

areas um this region here again

initiation so the plus one site and

again this shows 30 I said minus 25 30

somewhere around there you got the Tata

box and Upstream promoter elements more

of these elements you get the cat box

there's a GC Rich region GC Rich region

there's another cat box there um showing

these regions can

be um there in the promoter to help with

transcription

so in vetro experiments so in test tubes

have been done to try and figure out

what transcription factors bind where

and in what order in order for

transcription to start and I think I

have another slide here we'll get to

there we go to talk about this so UK

carotic initiation so how does the

initiation transcription take place in

UK carots the order of The Binding

then um discovered from inv vitro

transcription factors in vitro

transcription experiments where that D

needs to be involved first followed by a

which I haven't put here because a can

sometimes um be excluded

b r plas itself and

f e and H okay so there's an order to

these so d

first A and B rnaase 2 and F together

and E and then H okay so initiation of

RNA plas 2 transcription transcription

Factor

2D binds the hoges box or the Tata box

okay creating an initial committed

complex all right so the first event

that occurs transcription Factor

2D contains the Tata binding protein tbp

the Tata binding Protein Plus taffs TAF

which stand for Tata binding protein

Associated factors okay so trans Factor

2D is actually multiple proteins

together the Tata binding Protein Plus

taffs let's see if we

have something here this one doesn't

necessarily show it but it's showing the

first step transcription Factor 2D

binding there here you have trans

description Factor 2D is this big one

here and part of that is the Tata

binding protein actually all in blue

here is

um transcription Factor 2D in tata

binding protein is part of that uh

structure transcription Factor 2 B after

d binds tab box transcription Factor 2B

can

bind and recruits RNA plas to in trans

rtion factor to F forming What's called

the minimal

transcription um complex and again a is

involved um with B but it's it was found

to be dispensable in in vro

studies this complex this minimal

transcription

complex sorry um next after the minimal

transcription complex 2E and 2H combined

which complete the initiation complex

this initiation complex is sufficient

for low level of transcription or or

basil

transcription the start of transcription

is believed to be triggered by the

phosphorization of the carboxy terminal

domain of the RNA pmer beta subunit okay

RNA plas will sit down that uh region it

has at its carboxy end okay or carboxy

terminal domain it uh has a region that

needs to be phosphorated to sort of set

it in motion to make it go so let's take

a look at some figures if we can look

here so there's D binding and again B

comes in and a would be involved too

inside the cell which recruits F and the

plase here preinitiation or close

complex it's preinitiation complex there

then you get H and E sorry H and E then

gives the whole pre-initiation complex

and then it can start and so CDT of RNA

plas 2 it's our K boxal terminal domain

um you see the p4s here it gets

phosphorated and that's then what starts

the process this next one doesn't show

the start of the process but you got RNA

plase here D get all the other BF and

there's a in this particular picture and

this one showing um uh other proteins

involved so we'll talk about enhancers

and silencers later in the course but

they're talking about other proteins

that bind to other sequences in DNA that

can then help this process assemble more

quickly or or um start um faster so more

regulatory sequences that are helping

this event to

occur so then once it starts then we get

into the elongation stage and we'll come

back to this and jumping ahead um just a

little

bit all right so as I said there's these

other sequences that help regulate the

enhancers then the way they regulate

transcription they're located different

regions of the genome they can be

further Upstream than those promoter

elements they can be really far away

actually and they're specific in that

they bind activators which are proteins

then that help them bind the basil

transcriptional apparatus and they help

enhance transcription so their name is

aole right they are enhancers they

enhance transcription by binding

activator proteins silencers then can

help repress transcription or silence

transcription by binding repressors that

would then interact or not allow this um

Machinery to start transcribing uh one

of the very first enhancers was

discovered in yeast cells and it's

called the yeast uas or Upstream

activator sequence and it's a really

strong enhancer you put it in front of

most any Gene in an experimental setup

and it will really Drive um

transcription quite well all right so

let's get to we got to initiation C boox

terminal domain enhancers B activators

silencers by repressors so how does

elongation go well elongation occurs in

the same way in UK carots as it does in

Pro carots right that RNA pmer is going

to move along it's going to read the

template strand it's going to put in

these um nucleoside triphosphates cleave

off to the phosphates make those calent

bonds and

um all right so um we'll wrap up here

and then we'll start talking about

termination and the next

um lecture for this unit