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How is it possible that we share most of our DNA with a good among off animals yet we do not look like them ?
- Gene expression:
- A specific gene can be expressed at different rates, in different quantities, and at different times in organism that have very similar genomes and this leads to varying phenotype that we see.
- Gene expression at a different time and level lead to different phenotypes.
What qualities make RNA different from RNA?
- RNA is typically in a single stranded form
- It is made from a sugar ribose which has an hydroxyl on carbon number 2
- Has the ability to fold into specific shapes
- Uses the nucleotide Uracil instead of Thymine( A now bind to U)
- Has the ability to hybridize and become double-stranded
- RNA are recognized and destroyed while DNA is forever
- RNA can base pair internal with double strands are pairs of molecules paired together with RNA
- RNA is more like protein than it is like DNA
- Internal base pairing in RNA enable it to form different shapes, interact with different molecules,and form specific domains ( many of the same qualities of proteins)
Prokaryotic RNA polymerase
- Has four different subunits
- Attracted to promoter region of a gene
- requires a template of Double stranded DNA
- Requires all four activated precursor ( A,U, G, and C)
- Divalent metal ion ( Mg or manganese)
- Can start anywhere( de novo) and lay down the first nucleotide because it does not need a free 3' hydroxyl
Transcription= DNA to RNA
What are the challenges?
- Take the anti-sense strand and make RNA copy, then put the DNA back together.
- Separating exon from introns, but first have to copy both
- Have to start at the right place, copy the correct strand, go in the right direction, and know when to end.
- RNA is a temporary thing and will be removed anyway( no proofreading mechanism)
How do we know that RNA polymerase of starting at the right spot?
- There is a segment of DNA and it is labeled relative to the start site of transcription.
- The nucleotide at the start site of transcription is labeled +1. To the right is downstream and the numbers increase. Anything to the left is called upstream and the number decrease. Throughout the genome there is this numbering system associated with every gene.The Promoter is located in the upstream portion of the gene. And in the promoter are signals, specific sequences of DNA called DNA elements. Some of these elements have specific names and locations within the promoter. In bacteria, upstream of the start site centered about -10 is s DNA element witha sequence called the TATA box and this is one of the things that protein associated with transcription recognize and specifically bind to.
- TATA box is a DNA element that is in the promoter of both prokaryotes and eukaryotes.
DNA elements ( promoter signals)
- Elements that transcription proteins bind to:
- TATA box is a DNA element that is in the promoter of both prokaryotes and eukaryotes.
- Another important DNA element centered around -35 is called the Consensus sequence. There is also a consensus sequence with a small C.
- These two DNA elements are seen in bacteria
Eukaryote DNA elements
- They have a TATA box located at -25
- CAT box located at -75
- There are also enhancer sequences that can be found anywhere(upstream, downstream, in introns) and they are technically not apart of the promoter yet it effect transcription.
Break-down of RNA Polymerase
- It has a channel for nucleotides to feed into. Go into the active site in the center
- Allosteric region that can latch on to double stranded DNA as the helix moves into the active site. The direction of transcription is left to right. Enzyme latchs on tightly
- Region where the DNA reinhelle and feeds out
- Region called a Rutter. It is another allosteric region that is used to peel the newly form RNA molecule off of the DNA.
Looking back to make connections betweeen Prokaryotic and Eurkaryotic RNA polymerase
- Eukaryotic and Prokaryotic polymerase come from the same ancient ancestor double-psi barrel which is a cluster of beta sheets.
- This molecule consist of a antiparrallel beta sheet that form a barrel like structure and it cam together in a dimer and other lops of protein. All RNA polymerase evolved from this ancient core of the double sided barrel.
- Transcription starts from the left. There are multiple transcripts made at one.
- There are 9 types of RNA
Small nuclear RNAs
These RNA need to be spliced . RNA act enzymatically. help with splicing of mRNA
small cajal RNA, used to modify snoRNA and snRNAs
Seven steps to get a Primary Transcription:
Copy of genome that can be used to create protein?
( Prokaryptic RNA polymerase)
- There can be many DNA transcripts at one time.
- 1) There is a fifth sububnit for the RNA polymerase called the sigma factor and it is the one that reads the promoter and it come together with the other four sub units and this put together is called the HOLO enzyme.
- - what they do as a holo enzyme they scann along the double stranded genonme looking for the DNA elements upstream of the start site of transcription. The sigma factor has two binding sites and they can bind to the TATA box and the Consensus sequence at.
- By positioning the sigma factor on those two sites the, the RNA polymerase active site is positioned at the appropriate amount of enzyme downstream so that the RNA polymerase can start at plus one.
- One the sigma factor finds these two elements in the promoter, RNA polymerase is not in the active conformation as yet.
- It is important to have two active site of the sigma factor because this points the polymerase in the right direction.
- 2)The interaction of the sigma factor with the double stranded DNA leads it to start toi peel apart and reveal the template so that it is exposed to the active site of the polymerase the new transcripts can begin to be made.
- 3) a few nucletides are polymerized in the active site ( very slow process) , as long as the sigma factor is there, the polymerase is not yet committed to transcribing this gene.
- (ABORTIVE SYNTHESIS) during these first 8 or 10 nucleotides if the polymerase decides to abort the process it can because it is not committed. eventually the sigma factor lets go.
- 4) Eventually the sigma factor is released and when it lets go two conformation changes take place.First the RNA polymerase clamps down in the DNA tightly and is committed. Then, the transcript is starting to get longer, so it must be dissociated from the template so that the DNA can re hybridize .and thus the rutter changes its position so it can participate in the prying of the transcript bond to the template. The synthesis accelerates greatly
- 5) the signal for stopping is contained with the primary transcript.( in the case of prokaryotes) .Contained in the 3` end of the transcript are a bunch of GC base pairs and contained in these base pairs once they are synthesized will loop back around and hydrogen bond with each other and form a hair pin loop. As the hari pinb loop tries to come out past the rutter, it get hung up on it and tangled in it and the RNA polymerase get to this point of interaction between the rutter and the hair pin loop and it is forced open the rutter and this causes additional conformation change that causes the template to be released and then the few hydrogen bonds that are left between the template and the newly formed transcript are broken as well and the next step is that newly formed transcript being released. he RNA polyermase can be released and 6 or 7 sigma factors can got o find another sigma factor to synthesize another gene.
You do want all of your same genes synthesized all the time to the same degree.Thus there must be regulatory system ( in prokaryotes)
- Allow mutation in DNA element like the TATAAT box or the Concensus sequence. Thus the T and A swap out with G's and C's..etc.
- The more the DNA elements are changed and allowed to mutate the harder it is for the sigma factor to bind to the correct sequences. Thus, the sigma factor will not bind as well or as often in the promoters and this serves as a way of regulating how often that gene is activated.
- Another, way to regulate the gene expression is to put more spacing in between the DNA elements. There is an optimal number of nucleotides that belong in between them therefore by increasing them the rate of gene expression can be regulated.This affects the Sigma factor and causes it to have to stretch thus it will not want to express that gene as often. You can also remove nucleotides from in between these DNA elements and cause the sigma element to have to squeeze itself in.
- REGULATION BASICALLY CONTROLLED IN PROKARYOTES BY EITHER CHANGING THE NUCLEOTIDES IN OR THE SPACING BETWEEN THE DNA ELEMENTS.
- Plus, there are two elements because this tells the RNA polymerase which direction to go.
Why two DNA elements.
- Direct the DNA polymerase in the correct direction
- get the polymerase oriented on the correct template and position.
Eukaryotes RNA polymerase
- There are three RNA polymerases in Eukaryotes. Focusing on RNA polymerase.
- Eurkaryotic RNA polymerase is a very small portion of all the proteins that need to bind at the start site of transcription for eukarytoes. ( There are like 100-150 proteins needs to start at the start site of transcription)
RNA polymerase in prokaryotes
- TFII( Transcription factor two ).
- The first to bind when we want to transcribe a gene is TFIIb and one of the sububnits of this transcription factor is TBP ( TATA box binding protein). IT has a binding site for the TATA box sequence. The interactions that hold the bond of the TBP domain and the TATA box are weak and then this takes the DNA and puts stress on it by bending it and this makes it easier to peel it apart as well as attracts other factor.
- The second thing that binds is TFIIB, then RNA polymerase.
- CTD( carboxy terminal domain) - domain at the head of RNA polymerase. And in this is 52 repeats of the same seven amino acids . And in these repeats are serines that can be phosphorylated. TFIIH phosphorylates the serine in this RNA polymerase.
- Two specific activities:
- 1) works as a helicase and is responsible for prying about the DNA .
- 2) Another one of its subunits is a kinase.
A stretch of DNA that has a introns, extrons, and a promoter that codes for the formation of a specific protein.
RNA polymerase (Allosteric protein)
- There is a concensus sequence around the start site of transcription called INR( the concensus sequence) at +1
- IN DPE there is anotherr DNA element called DPE that is around +30.
The activator protein/ enhancer proteins
- Helps to bring everything together " the butterfly net"
- this binds to enhancer sites and due to the flexibility of DNA. There is also a mediator binding protein .
- Activator protein ( bind to enhancer sites that are close to the start site for transcription and can flip around due tot he flexibility of the DNA to the start site) then binds to the mediator protein that has binding site for all the proteins that are necessary for transcription.
Chromatin remodeling complexes or histone modifying enzymes.
can be apart of the mediator protein if there are proteins packed away that need to be transcribed.
Process of coping a segment of DNA into RNA.
In Prokarytoic Transcription
- There is no nucleus, thus the DNA is already floating around in the cell. The circular genome is free floating. Transcription occurs in the MRNA because it is still the link between DNA and protein. These mRNA as they are transcribed are ready for translation no further modification needs to occur just need the ribosome
- All the proteins necessary for a particular function are syhthsized together because the prokaryptic cell synthesis multiple transcripts in the same primary transcript
- Prokaryotic transcript have 3 phosphates at the 5` end
- They put multiple genes in a single transcript that can be later cleaved into individual proteins. All the proteins for a specific function can be synthesized together this way.
- DNA and MRNA are stuck in the nucleus . Thus we have to get out of thennuckleus, but you a=cannot do so as a primary RNA transcript. There nee to be multiple modifications that take place before eukaryotic RNA can leave the nucleus.
- First of all you are transcribed with the introns which do not code fo anything for the protein. The exons need to be connected and the introns need to be removed. ( splicing). Second, we have two modification to either end of the Mrna. If these modifications are not made the mrna will not be recognized bby the cell and will not be able to leave the nucleus.
Eukaryotic : Modification on the mRNA
- 1) the 5` end has to have a cap ( chemical modification). The 5` end modification happens first because this is the part that is synthesized first.
- As the message is transcribed there is an exon intron junction where the introns can be spliced out as they are bring made.
- 2)POly A tail is added to the 3` end and it is about 200-250 A residues. The poly A tail is needed for the MRNA to-get outside of the nucleus, you are not recognized by the ribosome as a message and it will not translate it otherwise, and finally the lifespan of a MRNA depends on the lengght of the POly A tail. Every time that a MRNA is translated, some of the A's are lost off of the poly A tail. Not having a poly A tail will ultimately lead to a message being recycled because it is not recognized as an MRNA any more.
The process of linking exons together and removing introns when during transcription.
The 5` cap
- Need to have a residue of 7 methyl guanosine in order to be recognized as a MRNA. A G residue. Theses guanosines are covalently linked via a triphosphate bridge connecting it to the 5` end in a unique 5` to 5` connection.Carbon # 5 of the cap is linked to carbon # 5 of the MRNA . this unique bond. ( called a 5` to 5` triphosphat bridge)
- This is what make MRNA stand out.
- How does this happen?
- the Carboxyl terminal domain from the RNA polymerase. All the phosphates added to the serine . Phospharylated serine of the tail attract proteins that do the splicing, capping, and the the one that add the poly A tails.
- They will all bind to and interact with this region of the polymerase. This when the RNA polymerase gets to transcribing and the 5` end is beginning to come out they will be in the position to interact with the transcript on the 5` end and make the appropriate modifications.
Step by step addition of the 5` cap
- 1)The phosphatase take the existing phosphates that are at the 5` end and removes one of them.Happen on Nascent( new) transcript.
- 2)Guanyl transferase -grabs a GTP and removes two of the phosphate and add the 3rd phosphate to the 5`end in the 5` to 5` linkage.
- 3) Methylase to methlate the G residue from the GTP . This happens atleast once on some caps but can happen more than once.
- Now the massage is capped.
- There are proteins that bind to the cap
Removing the introns during transcription
- We always have a 3` end of the first exon being linking to the 5` end of the next exon.
- There are signals within the transcript that signal where this junction point is. in thew intron somwehere is abn A reisdue and that A residue is surrounded by a specific group of neucleotides,. This A is unique because it can become acivated and the tyoe if activatioin that it requires is it that it is going to have to need to be able to participate in the breakage of the RNa molecule backbone at the first exon intron boundary, then we are going to link that breakage to the A and we are going to form a lasso ( lariat)
- When the lariat forms there now a a free 3` oh at the end of the exon and it needs to be brought over and once again we need to break the backbone at the junction of the lariat and the exon it is attach to . When the 3` Oh attached this eexon to join together the intron with it loop will be removed.
- The key to the process is the activation of that A .
- regulate the binding of actin and myosin.
- tropomysosin is also expressed in fibroblast and the brain along with striated and smooth muscle.
- Why do we need in them in fibroblasts ?
- actin makes up the cytoskeleton and tropomysosin helps to regulate cell movement and the growth and maintenance of the cytoskeleton. There is other cells beside muscle cells that might be appropriate for it to be expressed.
- The tropomyosin used in these different organelles are not the same type all across.
- Iof we were to transcribe all the trpomysoin we woulc have a certain primary transcript. But this doesn't happen. Not all the exons are always used in all the transcript. We have one gene but many different gene products and therefore five slightly different protein produced from the same gene.
In different tissues and in different cell types, the same gene can be used to produce different produces, based on the alternative linking of the exons. some exons gets left out, others get linked together and you end up with a different product in different cells.