Download Nabil Bashir 10-21

Hareth Inaya
RNA transcription
Nabil Bashir
10-21-2015
http://1drv.ms/1OJ4PZF
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First
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WARNING :
# before you start with this script you have to know that there is a lot of
explaining and clarifications that Im going to write
# SO please don’t make a shocked scared face .. Just relax , try to read ,
and understand
# once you understand you will automatically know what to memorize
# when you see this symbol ☯ it means the thing you are reading is not for
memorizing the doctor doesn’t mention it so its just for understanding
✄✄✄✄✄✄✄✄✄✄✄✄✄✄✄✄✄✄✄✄✄✄
♦ In this lecture we will learn about making and generating RNA via
what we call “ RNA transcription “
- it's basically copying one strand of the 2 DNA strands
- this process is one of three steps for producing a protein
- the sequence of the gene ( in the DNA ) determines what kind of
protein will be produced
*what we studied in the previous lecture was a very important concept
in making RNA, it's the start and the first step “the promoter “
* Promoter is simply :
1- a region of DNA that initiates transcription, the region that the
enzymes and other factors will plan their RNA transcription process
2- region in the DNA located at the left end of the DNA ( upstream on the
DNA I mean toward the 5’ end of the DNA )
3- so it’s a sequence of nucleotides , varies in its length , can reach up to
1000 base pair long
4- the boss of this site that will bind to it and start the whole thing is
‘ RNA polymerase‘
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♦ there are many elements ( regions ) in this promoter not all these
regions are for the enzymes to bind … there is a specific region for the
binding that we will mention.
what we need to know is 2 important regions : -10 region & -35 region
✿ -10 region :
1- first of all, what we are talking about is a region not a single
nucleotide .. its 6 base pair long ( ‫يعني بتتكون‬
Note:
‫ نكلوتايدز‬6 ‫ ) هايالمنطقة من‬and we call this region “
• all the sequences located at the left
TATA box “
of +1 is considered as a promoter
☯
2- the sequence in this region is the same in
almost at all the genes so we call it ‘
consensus sequence ‘ .. this sequence is :
TATAAT
• these 2 regions (-10 & -35) that
contain specific sequences called “ core
promoter “
•we call the -10 region in prokaryotes
“ pribnow box “
✿ -35 region : the same as -10 , it's 6 base-pair long , almost all the
genes have this specific region with the specific sequence so its called
“ consensus sequence “ .. this sequence is : TTGACA
**so what is the job of these sequences and where does the RNA
polymerase bind ?
- when RNA polymerase comes to the DNA for the transcription process
it needs to know the starting point.. from where should it initiate this
process ,,
and as we have mentioned , it’s the promoter’s job BUT it needs to know
where is the promoter !! and that’s the -35 job .. it knows where the
promoter is from this region then it binds to it .
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- after it binds to the -35 it receives a signal from -10 region , this signal
is needed so that RNA polymerase can fix and unwind the DNA strand .
##:RNA polymerase recognizes the -35 region and start to slide toward
the -10 region .. at -10 region it starts to unwind the DNA double strand
•• so -35 is to make the RNA polymerase recognize
the promoter , -10 sends a signal to fix it and
start unwinding
♦NOTE : RNA
polymerase starts the
transcription from +1
❅heat shock promoter :
- not all promoters have a similar -10 & -35 regions .. some of promoters
have a different specific sequence for a specific job
- Like the heat shock promoter that exist in what we call a heat shock
gene , the cell use it to respond to certain external or environmental
stimulus
- so we can say that these genes that contain these promoters are for
adaptive mechanisms .
• when you expose the cell to high temperature that will denature the
proteins inside that cell BY “ misfolding “ it
( fig. 1 )
•• so if we take e-coli or us as an example
cause it has the same mechanism ..
when our temperature is raised ( by fever)
there are heat shock factors in the cells ( the doctor said they are
specific proteins ) ,,once they receive that stimulus ( high
temperature)they go to what's called heat shock genes and bind to its
promoter ( heat shock promoter ) ,,,
then they activate those genes >> they will be expressed .. I mean
transcribed and translated >> then the product will be PROTEINS ..
they will protect the other proteins BY maintaining its proper folding
inside the cell ...
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♦NOTES :
‫بروتينات بننتجها علشان تحميلنا بروتينات ثانية ضعيفة ممكن تخرب قدام المؤثر الخارجي‬
‫تبعنا وبالمثال الي عنا اخدنا الحرارة‬
** if the cells don’t do this, all of our proteins will be denatured and we
will not be able to survive
**1 promoter could regulate more than one gene and those genes will
produce proteins that are correlated in function ( a student asked this
question to the doctor ) ♠as we see in the figure ,, the -10 & -35 regions
with different
sequence than the original
❅Nitrogen-starvation promoter :
- The same idea as heat shock promoter ,, so it's another example for
the promoters that differ in the -10 & -35 regions
- this promoter also have a specific job on certain conditions ,, here the
condition is : lack of nitrogen in the cells
•• once the bacteria finds no nitrogen source some factors ( proteins)
will bind to this sequence promoter in that certain gene >> and stimulate
transcription of the gene>> then translation >> that will produce
enzymes that are important to synthesize amino acids as nitrogen
source . and that’s all what the doctor said about this topic
♦ NOTE : these 2 genes that we talked about will be activated ( when
they are needed ) in specific cells or specific tissues not all tissues ,,
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◘ Transcription bubble :
* Template strand : the one that’s being copied
* coding strand : the one that’s out and being copied .
it's called ’ coding ‘ cause this strand will have the same
sequence as the RNA that we are making but U will be
instead of T
*memorize the keywords in this picture
• this bubble is a molecular
structure that occurs during the
transcription of DNA when a
limited portion of the DNA double strand is unwound
•• this yellow thing is the RNA polymerase molecule ..very big and
complex molecule
••• we mentioned before that this RNA polymerase is composed of
many subunits including ”sigma protein“ ,,, this protein is very
important to recognize the promoter region
•••• with out the sigma protein ( or if something happened to it ) RNA
polymerase will be unable to bind Without Sigma No transcription
♦lets make this together : transcription have 3 phases : initiation
phase,elongation phase and termination phase
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1✿ initiation phase :
• we actually talked about the first step in this phase .. when the sigma
protein of the RNA polymerase finds the promoter ( at -35 ) and binds to
it , this is the first step
•• after that ,RNA polymerase slides toward the -10 region and binds
more tightly then starts to unwind
RNA-DNA hybrid helix: is the pairing between the
the double helix strand of the
new mRNA and the template strand of the DNA . This
DNA
hybrid consists of the most recently added nucleotides of
••• RNA polymerase will
polymerize about 5 to 10 nucleotides
the RNA transcript, complementary base-paired to the
template strand
♦ Notes :
1- remember : RNA polymerase start copying at +1
2- polymerization of 5 to 10 nucleotide plus separation of the 2 strands
and formation of a small piece of RNA in the hybrid with the DNA ,,
this complex at a whole is called the “ open complex “
3- the end of the initiation phase is when the open complex is produced
4- if they ask a question : whats the initiation phase ?
- simply : the open complex
2✿Elongation phase :
• once the polymerization of the first 5 to 10 nucleotides, the sigma
factor will leave, then the elongation phase STAR
•• RNA polymerase will continue copying and making the RNA by adding
nucleotide ( ‫بنضل نضيفوحتى حدمعين‬Rna‫ عملية تطويل لل‬: )
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••• it has little proofreading ( error-correcting processes ) but not as
powerful as the DNA polymerase ,, WHY ???
- the cell does not need a powerful proofreading for RNA polymerase
because the mRNA molecule that’s formed have a very short life it will
be used once or twice, then it will be degraded .. not like DNA replication
,, DNA replication must have no errors , powerful proofreading
♦ Note :
1- the DNA-RNA hybrid is double stranded , but when its transcribed its
single stranded RNA .. how is that ?
- this hybrid formation is temporary and as it proceeds 5’ to 3’ ,,
note that the 5’ end of the RNA leaves the complex so the tail is outside
the RNA polymerase and only about 20 nucleotide are in hybrid with the
template
2- as the enzyme move to the right ( 5’ >> 3’ ) it unwinds more of the
DNA and the mRNA that we are making will get longer ,,
at the same time there will be rewinding the previous part of the DNA
back to the original status
3- the RNA-DNA hybrid rotate during elongation
4- PPP on the 5’ on mRNA is triphosphate
❅now before we talk about the last phase ,, there is a drug called
Rifampicin
** this rifampicin is an
antibiotic that will prevent
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RNA polymerase from transferring from the initiation phase to the
elongation phase so It will stop transcription
*and that's what you have to memorize*
3✿ termination phase :
There are 2 types of Terminaition :1) Termination signal by Stem loop
2) Rho Protein Termination .
1 ○ Termination by Stem loop :
• the figure above is RNA .. what we want from this phase is to remove
RNA polymerase from the transcription bubble once RNA polymerase is
lifted up every thing will be terminated
•• now what happens is that in some genes there are what’s called
“palindromic sequences “ these sequences are
○ Note :
- stem loop = hairpin
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complementary to each other within the DNA single strand .. they will
base pair with each other and form a double strand
••• so it acts like a jack,,
" ‫ نفس فكرة اخونا" الستم لووب‬.. ‫االشي الي بنرفع في السيارة لم يبنشر العجل‬
‫بتيجي ترفع السيارة علشان تشيل االنزيم فبالتالي بنشال ال" ار ان ايه " وبطيح وبتخلص العملية‬
•••• this kind of termination is called protein independent termination
OR sequence dependent termination because it doesn’t depend on any
sort of protein
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○ After that the doctor has mentioned this part :
There are some compounds in the cell if they are found abundantly
( ‫ )بكثرة‬they will stop the transcription for their synthesis ,,
( this is not negative feedback .. this is another different mechanism )
and if they are very low in concentration( then the cell needs them)
they will help not to form this stem loop and thus transcription for genes
that synthesize these compounds will go on so more synthesis of that
compound.
2 ○ Rho protein Termination :
• there is protein factor called RHO.
••This protein is an ATP-dependent helicase that binds the nascent RNA
chain and pulls it away from RNA polymerase and the DNA template.
•••it breaks RNA-DNA hybrid helix by its Helicase activity.
••••The signal that make Rho protein come is : sequences rich in Gs and
Cs. but Why? Because richness in Gs and Cs will slow the RNA
polymerase from polymerizing it and stops , why is it stopping there ?
-Because Rho is climbing and when it reach to top or RNA it will left it up
and terminate transcription.
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♦ Note :
- Rho factor is Hexamer has ATPase & Helicase activity.
○ “Some Antibiotics inhibit Transcription”
- Rifampicin and is antibiotic that inhibit transcription in prokaryotes.
-Actinomycin is antibiotic that inhibit transcription in
prokaryotes and eukaryotes.
1) Rifampicin specifically inhibit the Initiation of RNA synthesis NOT
ELONGATION à Rifampicin interferes with the formation of the first few
phosphodiester bonds in the RNA chain à it prevents transfer from
initiation phase to elongation phase.
2) Actinomycin D binds tightly and specifically to double-helical DNA
à prevents it from being an effective template for RNA synthesis.
♦tRNA and rRNA Processing in prokaryotes:
-In prokaryotes, messenger RNA molecules undergo little or no
modification after synthesis by RNA polymerase.
Indeed, many mRNA molecules are translated while they are being
transcribed. In contrast, transfer RNA and ribosomal RNA molecules are
generated by cleavage and other modifications of nascent RNA chains.
1) Cleavage :
-For example : in E.coli rRNA and tRNA are produced from one primary
RNA transcript that also contains space regions by cleavage.
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look to this figure :
-Cleavage of this transcript produces 5S, 16S, and 23S rRNA molecules
and a tRNA molecule , This cleavage is done by 2 enzymes:
1) Ribonuclease P à generates the correct 5’end of all tRNA molecules.
2) Ribonuclease III à cuts the 5s , 16s , 23S rRNA.
2) Modification of bases and ribose units of Ribosomal RNA:
-In prokaryotes : some rRNA are methylated.
-For example :uridylate residues are modified after transcription to form
ribothymidylate and pseudouridylate.
à this modification generate diversity.
Note: in prokaryotes there is:
1) tRNA is largely modified.
2) rRNA is little modified.
3) mRNA is very little modified
I apologize from my heart for the delay of this lecture but I swear I
was having some issues ,, my sincere apology
❀you to go and read script that Ammar Madkhana made
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❀if you have some time go and watch this video :
https://www.youtube.com/watch?v=9WNOZzO-NRU
❀ best wishes for all
‫واتمنى من كل قلب لكم النجاح‬.. ‫صر بذلنا ما بوسعنا لمساعدتكم‬
ٌ ‫سامحونا عالتق‬
Done by : HARETH INAYA ^^
Edited by : Layth H. Al-Zu'bi & Ahmad N.Al-Halawani
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