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The complexity of transcription through RNA polymerase enzymes and general transcription factors in eukaryotes

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The complexity of  transcription through RNA polymerase enzymes  and general transcription factors in eukaryotes





The Central Dogma of Molecular Biology: "DNA makes RNA makes protein" Here the process begins. Transcription factors assemble at a specific promoter region along the DNA. The length of DNA following the promoter is a gene and it contains the recipe for a protein. A mediator protein complex arrives carrying the enzyme RNA polymerase. It manoeuvres the RNA polymerase into place... inserting it with the help of other factors between the strands of the DNA double helix. The assembled collection of all these factors is referred to as the transcription initiation complex... and now it is ready to be activated. The initiation complex requires contact with activator proteins, which bind to specific sequences of DNA known as enhancer regions. These regions may be thousands of base pairs distant from the start of the gene. Contact between the activator proteins and the initiation-complex releases the copying mechanism. The RNA polymerase unzips a small portion of the DNA helix exposing the bases on each strand. Only one of the strands is copied. It acts as a template for the synthesis of an RNA molecule which is assembled one sub-unit at a time by matching the DNA letter code on the template strand. The sub-units can be seen here entering the enzyme through its intake hole and they are joined together to form the long messenger RNA chain snaking out of the top.

http://reasonandscience.heavenforum.org/t2036-the-complexity-of-transcription-through-rna-polymerase-enzymes-and-general-transcription-factors-in-eukaryotes#3451

The preinitiation complex (PIC) is a large complex of proteins that is necessary for the transcription of protein-coding genes in eukaryotes and archaea. 8







Transcription by RNA polymerase II (Pol II) is a finely tuned and multistep process of making RNA from a DNA template  that requires the concerted action of a large set of transcription factors.  RNA polymerase II transcription proceeds through multiple stages designated preinitiation, initiation, and elongation.  Several key factors are involved in this process. Including, DNA, transcription factors, RNA polymerase, and ATP. There are many more molecular machines involved in the process, namely additional proteins such as coactivators, chromatin remodelers, histone acetylases, deacetylases, kinases, and methylases etc..... The whole system is interdependent. The machinery itself is irreducible complex. DNA, transcription factors, RNA polymerase, and ATP must be present, otherwise transcription cannot occur. What came first, the TATA Box in the promoter region in DNA, or transcription factors, controlling the rate of transcription of genetic information from DNA to messenger RNA ? What use does one have without the other ? Both must have come into existence at the same time, since they are functionless, one without the other. And so the RNA polymerase machine as well, since the other two without it have no function either. That is a extremely sophisticated, interdependenet system that had to come into existence all at once. Thats best explained through a designer. this is a interdependent, highly coordinated complex system, where the single parts have no use, unless in conjunction with all other parts. This is one more prima facie example of intelligent design in molecular biology.

Furthermore, as Stephen Meyer explains in his book, Signature in the cell,

in both prokaryotes and eukaryotes, transcription constitutes a complex, functionally integrated process involving several specialized and individually necessary proteins. Yet production of each of these separate proteins is itself dependent on the very process of transcription that they make possible. To build RNA polymerase, for example, the cell must transcribe the genetic texts with the instructions for building RNA polymerase. Yet to transcribe this information requires RNA polymerase. The information necessary to manufacture RNA polymerase and all the other associated enzymes and protein cofactors of transcription is stored on the DNA template. But expressing that information on the DNA template for building the proteins of the transcription system requires most of the proteins of the transcription system.

What good is DNA for, if the transcription machinery is not in place in the cell to read the coded information stored in DNA ?  Must both not be in place and fully functional at the same time ? and what good is the transcription machinery good for without DNA to read the message ?


Discovery 7

In the 1950's, it was scientific belief that DNA was directly involved in protein synthesis, as the cellular function of RNA was not understood. However, based on the fact that reticulocytes and enucleated cells still produced proteins, some scientists deduced that DNA was not directly involved in protein synthesis. Polymerase phosphorylase was predicted to be responsible for cellular RNA synthesis (Losick et al 1976) until 1959 when Samuel B. Weiss and Leonard Gladstone first discovered DNA-dependent RNA polymerizing activity in the nuclear fraction of rat liver cells. Before then, active polymerase was always discovered in RNA extracts of bacterial RNA.

Weiss decided to run an experiment based on the assumption that RNA synthesis should be analogous to DNA synthesis (Losick et al 1976). He phosphorylated [32P]-labeled 5'CMP with ATP and crude brewer's yeast. The resultant [32P] was eluted on a Dowex-1-Cl column. A crude rat liver fraction isolated from the lower sedimenting fraction containing cell debris and nuclei showed incorporation of the [32P]-CTP into a TCA-precipitable fraction of RNA. Optimal incorporation of the labeled CTP occurred in the presence of other unlabeled ribonucleotide triphosphates as opposed to diphosphates. Addition of DNase slowed the incorporation, while addition of RNase significantly reduced the RNA chain length. However, Weiss and Gladstone did not indicate the presence of more than one class of RNA polymerase.

Later in 1969, Robert G. Roeder and William L Rutter separated and named the three mammalian RNA polymerases. They considered that there might be three different classes of RNA polymerases based on 3 assumptions:

1. Ribosomal RNA has a base composition different from other classes of RNA molecules.

2. Ribosomal genes are found in the nucleolus, while other classes of RNA molecules are dispersed in the nucleoplasm.

3. The regulation of ribosomal RNA synthesis and "DNA-like RNA," i.e. messenger RNA (mRNA) were independently regulated.

They isolated nuclei from rat liver cells. The RNA polymerase "activities" were dissolved and incompletely purified using sonication at a high ionic concentration, causing a high enzyme yield. They lowered the ionic concentration removed chromatin from the solution using centrifugation and ammonium sulfate fractionation. The enzymes were wholly purified using DEAE-Sephadex chromatography. Each polymerase was isolated and named according to its ammonium sulfate elution concentration. RNA polymerase I was isolated at the highest elution concentration. Polymerase II was isolated at medium salt concentration and polymerase III was isolated at the lowest concentration (Young 1991; Roeder and Rutter 1970).

The polymerases maintained functionally discrete properties after freezing, thawing, dilution, reconcentration, and second chromatographic purification. Polymerase II was characterized as easily soluble and found primarily in the nucleoplasm. Also, high ionic strength of Mn2+ increased mRNA synthesis and this activity was in the nucleoplasm. RNA polymerase I was localized in the nucleolus, while the functions of RNA polymerase III had not been characterized yet. In a subsequent experiment, the dynamic duo confirmed different activities for polymerases I and II at different stages of sea urchin embryo development. They found that during early cell division (blastula and postgastrula stages) synthesis of rRNA remains constant per cell attributed to the activity of RNA polymerase I, while mRNA synthesis increased in the cell. Thus, they confirmed that RNA polymerase II was responsible for the synthesis of "DNA-like RNA."





Transcription begins in the nucleus of the cell :



Many different types of chemical reactions are required to produce a properly folded protein from the information contained in a gene, namely



INITIATION OF TRANSCRIPTION

CAPPING,  ELONGATION,  SPLICING

CLEAVAGE,POLYADENYLATION AND TERMINATION

EXPORT FROM THE NUCLEUS TO THE CYTOSOL

INITIATION OF PROTEIN SYNTHESIS (TRANSLATION)

COMPLETION OF PROTEIN SYNTHESIS AND PROTEIN FOLDING

PROTEIN DEGRADATION




Transcription :

1,2

Transcription is the process of making RNA from a DNA template. Several key factors are involved in this process. Including, DNA, transcription factors, RNA polymerase, and ATP.

This is a irreducible complex system. DNA, transcription factors, RNA polymerase, and ATP must be present, otherwise transcription cannot occur.

Transcription begins with a strand of DNA. It is divided into several important regions. The largest of these is the transcription unit. This portion of the DNA will be used to produce RNA. Upstream of the transcription unit is the TATA box. An enhancer region may also be involved.

Several complexes, known as transcription factors, are required for successful transcription. The first is TFIID, the largest of the general factors. A component of this factor, TBP, binds to the DNA using the TATA box to position TFIID near the transcription initiation site. Other transcription factors, including TFIIA and TFIIB, then attach.

These complexes prepare the DNA for the successful binding of RNA polymerase. One RNA polymerase is bound, other transcription factors complete the mature transcription complex.

Now, energy must be added to the system for transcription to begin. This energy is provided by the reduction of ATP into ADP and Pi.

RNA polymerase then synthesizes an RNA template from the strand of DNA. Most factors are released after transcription begins. When the end of the transcription unit is reached, the RNA polymerase dissociates, and the newly formed strand of RNA is released.


All the parts must come into existence at the same time, one has no function without the others. Also the whole sequence of events must be coordinated, and all parts must fit precisely together. There is no feasable mechanism producing this complex system randomly and in a stepwise fashion. Evolution is not a option, since transcription is required to make proteins, which are required to make replication work, which is essential upon which evolution works.

In contrast to bacteria, which contain a single type of RNA polymerase, eucaryotic nuclei have three: RNA polymerase I, RNA polymerase II, and RNA polymerase
III.
RNA polymerase II transcribes most genes, including all those that encode proteins.

Lets concentrate just on RNA polymerase II

There are several important differences in the way in which the bacterial and eucaryotic enzymes function.

1.While bacterial RNA polymerase requires only a single additional protein (s factor) for transcription initiation to occur, eucaryotic RNA polymerases require many additional proteins.

2. Eucaryotic transcription initiation must deal with the packing of DNA into nucleosomes* and higher-order forms of chromatin structure, features absent from bacterial chromosomes.
3

Initiation of transcription of a eucaryotic gene by RNA polymerase II

To begin transcription, RNA polymerase requires several general transcription factors.The general transcription factors help to position eucaryotic RNA polymerase correctly at the promoter aid in pulling apart the two strands of DNA to allow transcription to begin, and release RNA polymerase from the promoter into the elongation mode once transcription has begun. The proteins are “general” because they are needed at nearly all promoters used by RNA polymerase II; consisting of a set of interacting proteins, they are designated as TFII (for transcription factor for polymerase II), and are denoted arbitrarily as TFIIB, TFIID, and so on.

Promoters contain specific DNA sequences such as response elements that provide a secure initial binding site for RNA polymerase and for proteins called transcription factors that recruit RNA polymerase. 4

In a broad sense, the eucaryotic general transcription factors carry out functions equivalent to those of the s factor in bacteria; indeed, portions of TFIIF
have the same three-dimensional structure as the equivalent portions of s.




The above image illustrates the key elements of a typical eukaryotic gene. Transcription begins at the transcription start site and proceeds in the direction of the red arrow stopping at the termination site. This transcribed region will become pre-mRNA which includes introns, exons, a 5’ (read: "five prime") untranslated region (UTR) and a 3’ UTR. The initiator (start) codon and the terminator (stop) codon mark the limits of the protein coding sequence.

Following table summarizes their activities :



The assembly process begins when the general transcription factor TFIID binds to a short double-helical DNA sequence primarily composed of T and A nucleotides. For this reason, this sequence is known as the TATA sequence, or TATA box, and the subunit of TFIID that recognizes it is called TBP (for TATAbinding protein). The TATA box is typically located 25 nucleotides upstream from the transcription start site. It is not the only DNA sequence that signals the start of transcription , but for most polymerase II promoters it is the most important. The general transcription factors help to position eucaryotic RNA polymerase correctly at the promoter.

The binding of TFIID causes a large distortion in the DNA of the TATA box . This distortion is thought to serve as a physical landmark for the location of an active promoter in the midst of a very large genome, and it brings DNA sequences on both sides of the distortion together to allow for subsequent protein assembly steps. Other factors then assemble, along with RNA polymerase II, to form a complete transcription initiation complex TFIIH. Consisting of 9 subunits, it is nearly as large as RNA polymerase II itself
and, as we shall see shortly, performs several enzymatic steps needed for the initiation of transcription.

After forming a transcription initiation complex on the promoter DNA, RNA polymerase II must gain access to the template strand at the transcription start point. TFIIH, which contains a DNA helicase as one of its subunits, makes this step possible by hydrolyzing ATP and unwinding the DNA, thereby exposing the template strand. Next, RNA polymerase II, like the bacterial polymerase, remains at the promoter synthesizing short lengths of RNA until it undergoes a series of conformational changes that allow it to move away from the promoter and enter the elongation phase of transcription. A key step in this transition is the addition of phosphate groups to the “tail” of the RNA polymerase (known as the CTD or C-terminal domain). In humans, the CTD consists of 52 tandem repeats of a seven-amino-acid sequence, which extend from the RNA polymerase core structure.During transcription initiation, the serine located at the fifth position in the repeat sequence (Ser5) is phosphorylated by TFIIH, which contains a protein kinase in another of its subunits .

The polymerase can then disengage from the cluster of general transcription factors. During this process, it undergoes a series of conformational changes that tighten its interaction with DNA, and it acquires new proteins that allow it to transcribe for long distances, and in some cases for many hours, without dissociating from DNA. Once the polymerase II has begun elongating the RNA transcript, most of the general transcription factors are released from the DNA so that they are available to initiate another round of transcription with a new RNA polymerase molecule. As we see shortly, the phosphorylation of the tail of RNA polymerase II also causes components of the RNA-processing machinery to load onto the polymerase and thus be positioned to modify the newly transcribed RNA as it emerges from the polymerase.

Polymerase II Also Requires Activator, Mediator, and Chromatin-Modifying Proteins

DNA in eucaryotic cells is packaged into nucleosomes, which are further arranged in higher-order chromatin structures. As a result, transcription initiation in a eucaryotic cell is
more complex and requires even more proteins than it does on purified DNA. First, gene regulatory proteins known as transcriptional activators must bind to specific sequences in DNA and help to attract RNA polymerase II to the start point of transcription.



For RNA polymerase to successfully bind to a eukaryotic promoter and initiate transcription, a set of proteins called transcription factors must first assemble on the promoter.
The assembly process begins upstream from the transcription start site, where proteins called basal factors bind to a short TATA sequence in the promoter.
Other basal factor proteins then bind, eventually forming a full transcription factor complex able to capture the RNA polymerase.
Basal factors are essential for transcription but cannot by themselves increase or decrease its rate. A second set of transcription factors called coactivators link the basal factors with a third set of transcription factor proteins called activators.
Activators are regulatory proteins that bind to sequences on DNA called enhancers. Enhancers are located at sites that are distant from the promoter.
The interaction of activator proteins with transcription factor subunits increases the rate of transcription.
Many enhancers, scattered around the chromosome, can bind different activators, which provide a variety of responses to various signals.
When a second kind of regulatory protein called a repressor binds to a "silencer" sequence located adjacent to or overlapping an enhancer sequence, the corresponding activator is no longer able to bind to the DNA.









The presence of activators on DNA is required for transcription initiation in a eucaryotic cell. Second, eucaryotic transcription initiation in vivo requires the presence of a protein complex known as Mediator, which allows the activator proteins to communicate properly with the polymerase II and with the general transcription factors. Finally, transcription initiation in a eucaryotic cell typically requires the local recruitment of chromatin modifying enzymes, including chromatin remodeling complexes and histonemodifying enzymes.

Many proteins (well over 100 individual subunits) must assemble at the start point of transcription to initiate transcription in a eucaryotic cell. The order of assembly of these proteins does not seem to follow a prescribed pathway; rather, the order differs from gene to gene. Indeed, some of these different protein complexes may interact with each other away from the DNA and be brought to DNA as preformed subassemblies.



1.Transcription begins with a strand of DNA. This simplified DNA model has been color-coded to show regions with important roles in transcription.



2.The largest key region is the transcription unit.



3.Upstream of the transcription unit is the TATA-box, a smaller section that helps to position the complexes involved in transcription.The assembly process begins when the general transcription factor TFIID binds to a short double-helical DNA sequence primarily composed of T and A nucleotides. For this reason, this sequence is known as the TATA sequence, or TATA box



4.The final high-lighted region is the enhancer.




5. TFIID, a general transcription factor, is shown approaching the strand of DNA.



6. TFIID is the largest of the general transcription factors involved in eukaryotic transcription. The yellow part of the complex is called TBP.  (for TATAbinding protein)



7. TBP (yellow) binds to the DNA, using the TATA-box to position itself near the iniation site of transcription.Through its subunit
TBP, TFIID recognizes and binds the TATA box, which then enables the adjacent binding of TFIIB





8. When the TBP portion of the TFIID molecule attaches to the TATA-box, its shape causes the DNA to bend.




9.Two smaller general transcription factors are shown coming into view: TFIIA (orange) and TFIIB (red).



10. TFIIA binds to the TATA-box near TFIID.



11. TFIIB approaches the TATA-box. The Pol II complex is being assembled in the distance.



12.TFIIB binds to the TATA-box and TFIID. It is thought to help the Pol II complex bind correctly.



13. The Pol II complex has been assembled and is approaching the start site for transcription.



14. Aided by the general transcription factors already in place, Pol II binds to the DNA strand at the start site for transcription. TFIIE is show approaching from the right.



15. Additional factors must still bind to the complex in order to start transcription. Here, TFIIE has already bound (olive green) and TFIIH (red) is preparing to do the same.



16. Once all of the general transcription factors are bound, energy, in the form of ATP (blue/pink), is needed to activate the Pol II complex.



17. Once the ATP have been added, an 'eye' opens in the DNA giving access to the DNA template, and the creation of mRNA can begin.



18. Once mRNA elongation begins, TFIIE (olive green) and TFIIH (red) are released. Also released at this time are TFIIA and TFIIB (not shown).



19. TFIIE is shown moving into the distance, and the mRNA strand is elongating rapidly.



20. Elongation of the mRNA stops when the end of the transcription unit is reached.



21. As elongation stops, the DNA 'eye' closes.



22. The Pol II complex is now released from the DNA, along with the phosphates added to it by the ATP.



23.As the Pol II complex dissociates from the DNA, the mRNA strand is released. At this time, TFIID also unbinds from the DNA, allowing the DNA to return to its normal shape.



24.Transcription is now complete. An mRNA copy has been produced and is now ready to be moved outside of the nucleus and be used in the translation process.


Transcription preinitiation complex

The initial event of transcription of any gene requires the formation of a pre-initiation complex which associates with the gene’s promoter region. Once activation of this complex has occurred, the RNA polymerase leaves the promoter region and begins the process of elongation of nascent RNA chains. 4

RNA polymerase II (pol II) is capable of RNA synthesis but is unable to recognize a promoter or to initiate transcription. For these essential functions, a set of general transcription factors (GTFs)—termed TFIIB, -D, -E, -F, and -H—is required. 6

The minimal preinitiation complex (abbreviated PIC)  includes  six general transcription factors: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. Additional regulatory complexes (co-activators and chromatin-remodeling complexes) could also be components of the PIC.

Assembly

A classical view of PIC formation at the promoter involves the following steps:
TATA binding protein (TBP, a subunit of TFIID) binds the promoter, creating a sharp bend in the promoter DNA.
TBP-TFIIA interactions recruit TFIIA to the promoter.
TBP-TFIIB interactions recruit TFIIB to the promoter.
TFIIB-RNA polymerase II and TFIIB-TFIIF interactions recruit RNA polymerase II and TFIIF to the promoter.
TFIIE joins the growing complex and recruits TFIIH.
Subunits within TFIIH that have ATPase and helicase activity create negative superhelical tension in the DNA.
Negative superhelical tension causes approximately one turn of DNA to unwind and form the transcription bubble.
The template strand of the transcription bubble engages with the RNA polymerase II active site.
RNA synthesis begins.
After synthesis of ~10 nucleotides of RNA, and an obligatory phase of several abortive transcription cycles, RNA polymerase II escapes the promoter region to transcribe the remainder of the gene.
An alternative hypothesis of PIC assembly postulates the recruitment of a pre-assembled "RNA Polymerase II holoenzyme" directly to the promoter (composed of all, or nearly all GTFs and RNA polymerase II and regulatory complexes), in a manner similar to the bacterial RNA polymerase (RNAP).

Question : How could the proteins above involved in transcription preinitiantion arise by natural processes ? They are only functional if working as a whole in a highly coordentated manner ?! The above process could not arise in a stepwise manner, but works only if fully functioning right from the start. The process could not be result of evolution, since in order for evolution to work, transcription must already be in place ?
RNA polymerase III :

http://mol-biol4masters.masters.grkraj.org/html/Gene_Expression_II4-RNAP_III_Promoter.htm


* Nucleosomes: http://reasonandscience.heavenforum.org/t2017-the-nucleosomes-amazing-design#3392

1) https://www.youtube.com/watch?v=WsofH466lqk
2) http://vcell.ndsu.nodak.edu/animations/transcription/movie-flash.htm
3) http://reasonandscience.heavenforum.org/t2017-the-nucleosomes-amazing-design#3392
4) http://en.wikipedia.org/wiki/Promoter_%28genetics%29
5) http://www.biology.emory.edu/research/Lucchesi/html/research.html
6) http://www.sciencemag.org/content/342/6159/1238724
7) http://www.biochem.umd.edu/biochem/kahn/molmachines/newpolII/discovery.html
8 ) https://en.wikipedia.org/wiki/RNA_polymerase_II_holoenzyme



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2 TATA-binding protein on Thu Jun 04, 2015 8:49 pm

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The TATA-binding protein

TATA binding protein (TBP, a subunit of TFIID) binds the promoter, creating a sharp bend in the promoter DNA.



Introduction

The enzyme RNA polymerase performs the delicate task of unwinding the two strands of DNA and transcribing the genetic information into a strand of RNA. But how does it know where to start? Our cells contain 30,000 genes encoded in billions of nucleotides. For each gene, the cell must be able to start transcription at the right place and at the right time.

Getting Started

Specialized DNA sequences next to genes, called promoters, define the proper start site and direction for transcription. Promoters vary in sequence and location from organism to organism. In bacteria, typical promoters contain two regions that interact with the sigma subunit of their RNA polymerase. The sigma subunit binds to these DNA sequences, assists the start of transcription, and then detaches from the polymerase as it continues transcription through the gene. Our cells have a far more complex promoter system, using dozens of different proteins to ensure that the proper RNA polymerase is targeted to each gene. The TATA-binding protein is the central element of this system.

The TATA Box

Our protein-coding genes have a characteristic sequence of nucleotides, termed the TATA box, in front of the start site of transcription. The typical sequence is something like T-A-T-A-a/t-A-a/t, where a/t refers to positions that can be either A or T. Surprisingly many variations on this theme also work, and one of the challenges in the study of transcription is discovering why some sequences work and others don't. The TATA-binding protein (sometimes referred to as TBP) recognizes this TATA sequence and binds to it, creating a landmark that marks the start site of transcription. When the first structures of TATA-binding protein were determined, researchers discovered that TATA-binding protein is not gentle when it binds to DNA. Instead, it grabs the TATA sequence and bends it sharply, as seen in PDB entries 1ytb, 1tgh and 1cdw.

The TATA box-binding protein (TBP) is an essential component of the RNA polymerase II transcription apparatus in eukaryotic cells. 1

Transcription initiation is one of the key regulatory steps in the control of gene expression:  initiating transcription at the correct promoter at the correct time is essential for executing the correct biological process.  The transcription of eukaryotic genes requires one of three RNA polymerases: Pol I, Pol II, or Pol III.  However, none of these RNA polymerases can initiate transcription on its own, instead requiring the aid of different sets of transcription factors.  The TATA-box binding protein (TBP) is required for the accurate initiation of transcription by all three transcription factors from promoters with or without a TATA-box, associating with a variety of different transcription factors. 2

TBP associates with distinct sets of TBP-associated factors (TAF), which enable the accurate initiation of transcription using specific RNA polymerases.  For example, Pol II transcription can involve TFIID in which TBP associates with 13 to 14 different TAFs, or B-TFIID in which TBP associates with BTAF1. These associations can be highly specific.  For example, the association of TBP with an unprocessed form of TFIIA in the TBP-TFIIA-containing complex (TAC) is specific for undifferentiated stem cells.





1) http://www.ncbi.nlm.nih.gov/pubmed/10220372
2) https://www.ebi.ac.uk/interpro/potm/2005_7/Page1.htm

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Transcription factor II A  TFIIA   1

TBP-TFIIA interactions recruit TFIIA to the promoter



TFIIA interacts with the TBP subunit of TFIID and aids in the binding of TBP to TATA-box containing promoter DNA.  Interaction of TFIIA with TBP facilitates formation of and stabilizes the preinitiation complex. Interaction of TFIIA with TBP also results in the exclusion of negative (repressive) factors that might otherwise bind to TBP and interfere with PIC formation. TFIIA also acts as a coactivator for some transcriptional activators, assisting with their ability to increase, or activate, transcription.

TFIIA is thought to be the next protein to bind to the transcription-initiation complex.  It joins TFIID and TBP at the promoter, where footprinting indicates it covers base pairs from –80 to –17 from the start site for transcription (McClean, 1997).  The structure of this protein varies slightly from species to species; it has 2 very acidic sub-units in yeast, and 3 in drosophila and humans (Buratowski, 2001).  The Richmond lab, using x-ray crystallography, has determined the structure of a complex involving TFIIA at a resolution of 2.5 Angstroms.  It appears to have a boot-shaped core containing 2 separate parts: 1 contains a ß barrel, the other is a bundle of 4 alpha-helices (Richmond, 1999).



1) http://en.wikipedia.org/wiki/Transcription_factor_II_A



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Transcription factor II B    TFIIB   1

TBP-TFIIB interactions recruit TFIIB to the promoter

Transcription Factor II B (TFIIB) is a general transcription factor that is involved in the formation of the RNA polymerase II preinitiation complex and aids in stimulating transcription initiation. TFIIB is localised to the nucleus and provides a platform for PIC formation by binding and stabilising the DNA-TBP (TATA-binding protein) complex and by recruiting RNA polymerase II and other transcription factors. It is encoded by the TFIIB gene

TFIIB is thought to be the third complex to interact with the DNA promoter region.  A small protein, it contains only one subunit about 35-40 kD in size.  It contains a Zinc-finger domain at its N-terminus, where it binds to the DNA (Buratowski, 2001). It also binds directly to and stabilizes TBP binding to DNA by causing conformational changes in TFIID ’s binding cavity (Brody, 2001).

TFIIB’s position on the DNA was found using DNA footprinting, which indicated that it covers base pairs –80 to –17 and –10 to +10 (McClean, 1997).  Using electron microscopy, the Lawrence Berkeley National Laboratory learned its position in relation to TFIID, TFIIA , and DNA in the complex (Yarris, 1999).  
Through an interaction with TFIIF , TFIIB recruits RNA Polymerase II to the promoter site (Brody, 2001).  TFIIB also may be a site where regulatory factors can bind to either enhance or block the initiation of transcription (Buratowski, 1999). 2




1) http://en.wikipedia.org/wiki/Transcription_factor_II_B
2) http://www.biochem.umd.edu/biochem/kahn/molmachines/newpolII/TFIIB.html



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5 Transcription factor II D TFIID on Sat Jun 06, 2015 1:37 pm

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Transcription factor II D  TFIID

"TFIID is the first protein to bind to DNA during the formation of the pre-initiation transcription complex of RNA polymerase II (RNA Pol II). Binding of TFIID to the TATA box in the promoter region of the gene initiates the recruitment of other factors required for RNA Pol II to begin transcription. Some of the other recruited transcription factors include TFIIA, TFIIB, and TFIIF. Each of these transcription factors is formed from the interaction of many protein subunits, indicating that transcription is a heavily regulated process." 5

TFIID triggers pre-initiation complex formation, functions as a coactivator by interacting with transcriptional activators and reads epigenetic marks. TFIID is a megadalton-sized multiprotein complexcomposed of TATA-box-binding protein (TBP) and 13 TBP-associated factors (TAFs). Despite its crucial role, the detailed architecture and assembly mechanism of TFIID remain elusive. 6

Before the start of transcription, the transcription Factor II D (TFIID) complex binds to the TATA box in the core promoter of the gene. 1

TFIID is itself composed of several subunits called TATA-binding protein Associated Factors (TBP-associated factors, or TAFs). In a test tube, only TBP is necessary for transcription at promoters that contain a TATA box. TAFs, however, add promoter selectivity, especially if there is no TATA box sequence for TBP to bind to. TAFs are included in two distinct complexes, TFIID and B-TFIID. The TFIID complex is composed of TBP and more than eight TAFs. But, the majority of TBP is present in the B-TFIID complex, which is composed of TBP and TAFII170 (BTAF1) in a 1:1 ratio. TFIID and B-TFIID are not equivalent, since transcription reactions utilizing TFIID are responsive to gene specific transcription factors such as SP1, while reactions reconstituted with B-TFIID are not.
Subunits in the TFIID complex include:

TBP (TATA binding protein)
TAF1 (TAFII250)
TAF2 (CIF150)
TAF3 (TAFII140)
TAF4 (TAFII130/135)
TAF4B (TAFII105)
TAF5 (TAFII100)
TAF6 (TAFII70/80)
TAF7 (TAFII55)
TAF8 (TAFII43)
TAF9 (TAFII31/32)
TAF9B (TAFII31L)
TAF10 (TAFII30)
TAF11 (TAFII28)
TAF12 (TAFII20/15)
TAF13 (TAFII18)
TAF15 (TAFII68)

TFIID, the largest of the GTF’s, is believed to be the first to bind to the promoter region of DNA (Buratowski, 1999).  It comprises the large TATA box binding protein (TBP) along with up to 7 other polypeptide sub-units, containing “an HMG box, bromodomains, and a serine kinase” (Brody, 2001).  It also appears to have histone acetyltransferase (HAC) activity.  It has been found to exist, like a nucleosome, as a heteotetramer in solution, leading many to believe it has a histone-octamer structure (Brody, 2001). 2




The eukaryotic core promoter recognition complex was generally thought to play an essential but passive role in the regulation of gene expression. However, recent evidence now indicates that core promoter recognition complexes together with 'non-prototypical' subunits may have a vital regulatory function in driving cell-specific programmes of transcription during development. Furthermore, new roles for components of these complexes have been identified beyond development; for example, in mediating interactions with chromatin and in maintaining active gene expression across cell divisions. 4


1) http://en.wikipedia.org/wiki/Transcription_factor_II_D
2) http://www.biochem.umd.edu/biochem/kahn/molmachines/newpolII/TFIID.html
3) http://www.nature.com/nrg/journal/v11/n8/full/nrg2847.html#B12
4) http://www.nature.com/nrg/journal/v11/n8/full/nrg2847.html
5) http://en.wikiversity.org/wiki/General_transcription_factor_II_D
6) http://www.ncbi.nlm.nih.gov/pubmed/23292512



Last edited by Admin on Fri Jun 12, 2015 12:02 am; edited 2 times in total

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Transcription factor II E TFIIE

Transcription factor II E (TFIIE) is one of several general transcription factors that make up the RNA polymerase II preinitiation complex 1

The fifth factor to bind to this large complex is TFIIE, a protein with 1 large and 1 small sub-unit (56kD and 34kD in humans) (Brody, 2001).  This protein exists as a tetramer in solution (2 molecules of each sub-unit bind together).  The large sub-unit contains a Zinc-finger domain, a common DNA binding motif in proteins (Buratowski, 1999).  Through DNA footprinting experiments, it has been shown that TFIIE binds DNA between -80 and –30 base pairs from the start site (McClean, 1997).

 Its main role in the transcriptional complex is to recruit TFIIH to the site, then regulate the helicase and kinase activities of TFIIH.

TFIIE is also necessary for RNA Polymerase II to switch into elongation mode (Buratowski, 1999).   2




1) http://en.wikipedia.org/wiki/Transcription_factor_II_E
2) http://www.biochem.umd.edu/biochem/kahn/molmachines/newpolII/TFIIE.html

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7 Transcription factor II H TFIIH on Sat Jun 06, 2015 2:07 pm

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Transcription factor II H  TFIIH



TFIIH consists of ten subunits, 7 of which

XPD
XPB
p62
p52
p44
p34  
TTDA


form the core complex. The cyclin activating kinase-subcomplex

CDK7
MAT1
cyclin H


is linked to the core via the XPD protein
 Two of the subunits, ERCC2/XPD and ERCC3/XPB, have helicase and ATPase activities and help create the transcription bubble.

In a test tube these subunits are only required for transcription if the DNA template is not already denatured or if it is supercoiled.
Two other TFIIH subunits,

CDK7
cyclin H



phosphorylate serine amino acids on the RNA polymerase II C-terminal domain and possibly other proteins involved in the cell cycle. Next to a vital function in transcription initiation, TFIIH is also involved in nucleotide excision repair.
It is responsible for giving the 'go' signal which is why it is assembled last  1

TFII – H has nine subunits (Roeder, 1996).  Its primary function is to initiate transcription and repair DNA damage (Bradsher et al, 2000).  TFII-H can perform enzymatic activities, acting as a DNA helicase and an ATP kinase (Papavassiliou, 1997).  During transcription initiation, a stable preinitiation complex (PIC) is completed by the binding of TFII – H (Bradsher et al, 2000).  The two largest subunits of TFII-H, XPB and XPD, are responsible for performing enzymatic activities to initiate transcription by melting the promoter regions around the transcription origin (Winkler et al, 2000).  The melting of the promoter region is ATP-dependent; TFII-H is important since it is the only transcription factor that is capable of ATP kinase activity (Hampsey, 1998).  Moreover, TFII-H can phosphorylate the carboxyl terminal domain (CTD) of RNA pol II, facilitating transcription elongation (Winkler et al, 2000 and Weaver, 2002).

TFII –H can also repair damaged DNA by nucleotide excision repair (NER) (Hampsey, 1998 and Winkler et al, 2000).

 The helicase activities of XPB and XPD allow recognition and removal of DNA damage (Winkler et al, 2000).  Any mutations or defects in XPB and XPD can cause diseases such as xeroderma pigmentosum, Cockayne’s syndrome and trichothiodystrophy (Bradsher et al, 2000 and Winkler et al, 2000).   2


TFIIH is a multifunctional RNA polymerase II transcription factor that possesses DNA-dependent ATPase, DNA helicase, and protein kinase activities. Previous studies have established that TFIIH enters the preinitiation complex and fulfills a critical role in initiation by catalyzing ATP-dependent formation of the open complex prior to synthesis of the first phosphodiester bond of nascent transcripts. In this report, we present direct evidence that TFIIH also controls RNA polymerase II activity at a postinitiation stage of transcription, by preventing premature arrest by very early elongation complexes just prior to their transition to stably elongating complexes. Unexpectedly, we observe that TFIIH is capable of entering the transcription cycle not only during assembly of the preinitiation complex but also after initiation and synthesis of as many as four to six phosphodiester bonds. These findings shed new light on the role of TFIIH in initiation and promoter escape and reveal an unanticipated flexibility in the ability of TFIIH to interact with RNA polymerase II transcription intermediates prior to, during, and immediately after initiation. 3



Nucleotide excision repair 4

Nucleotide excision repair (NER) is a mechanism to recognize and repair bulky DNA damage caused by compounds, environmental carcinogens, and exposure to UV-light. In humans hereditary defects in the NER pathway are linked to at least three diseases: xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD). The repair of damaged DNA involves at least 30 polypeptides within two different sub-pathways of NER known as transcription-coupled repair (TCR-NER) and global genome repair (GGR-NER). TCR refers to the expedited repair of lesions located in the actively transcribed strand of genes by RNA polymerase II (RNAP II). In GGR-NER the first step of damage recognition involves XPC-hHR23B complex together with XPE complex (in prokaryotes, uvrAB complex). The following steps of GGR-NER and TCR-NER are similar.

Transcription coupled repair (TC-NER)

5

TC-NER initiates when RNA polymerase stalls at a lesion in DNA: the blocked RNA polymerase serves as a damage recognition signal

A simple way to envision the action of UvrD in prokaryotes to promote RNA polymerase backtracking is the train engine analogy, in which a locomotive (RNA polymerase) encounters a railroad track buckle (DNA damage) and stalls. A second locomotive (UvrD in blue) is positioned at the rear end of the first locomotive, and the UvrD throttle is engaged to quickly and efficiently drag the stalled RNA polymerase locomotive away from the track buckle, so the repair crew can fix it. In this scenario, UvrD utilizes its force-producing ATP hydrolysis to haul the large 5-subunit RNA polymerase complex of ~400 kiloDalton backward, overcoming the driving force of the elongating RNA polymerase to translocate again toward the damage and obstruct the buckle. Once the lesion is repaired, RNA synthesis can resume.  6



Researchers are beginning to appreciate a role for RNA polymerase beyond gene transcription.  7

Long known for its role in transcribing the genome’s code into messenger RNAs that can be translated into proteins, the enzyme RNA polymerase may also survey the genome for damage. That’s according to a study led by investigators at the New York University Langone Medical Center, which was published last month (January Cool in Nature. Biochemist Evgeny Nudler and his colleagues have described one way in which bacterial cells rely on RNA polymerase to start repairing DNA damage, which, the authors added, hint at pervasive transcription—the surprising revelation of noncoding RNA molecules and an axis of debate in molecular biology.

As RNA polymerases glide along strands of DNA transcribing them into new RNA molecules, the enzymes can at times get stuck when they encounter damage in the genome, like a zipper caught on a faulty tooth. “RNA polymerase gets stuck all the time,” explained Nudler. “It’s very picky.” While minor damage can cause the enzyme to merely pause briefly, major damage can stop it cold, disrupting gene expression and DNA replication.

One way to get RNA polymerase going again involves a molecule known as UvrD helicase. Through a series of in vitro and in vivo experiments, Nudler’s team showed how this molecule pulls the transcription enzyme back from the damage site by unwinding DNA strands. “Eight or 10 nucleotides back is enough to expose the lesion to repair enzymes,” Nudler said. The researchers also showed that UvrD recruits the repair team, which excises and replaces damaged DNA.

Though the study only focused on one type of DNA repair in E. coli, it’s a mechanism that appears to be conserved throughout the tree of life, said James Cleaver from the University of California, San Francisco, who studies DNA repair-related diseases and was not involved in the work.

“It’s very helpful to see the same backtracking mechanism,” which has been suggested in humans and other eukaryotes, Cleaver said. “In all the years of studying DNA repair, there are still details to unravel in E. coli, which is supposedly a simple little bug.”

In humans, RNA polymerase cofactors CSA and CSB, or the proteins XPC and XPE, perform the same function as UvrD, helping RNA polymerase backtrack along the genome to expose DNA damage, said Cleaver. People who have mutations in this pathway suffer from conditions called xerodema pigmentosum or Cockayne syndrome, in which their cells cannot fix damage caused by ultraviolet radiation or other mutagens. Exposure to sunlight in xeroderma pigmentosum can cause skin and eye damage that greatly raises the risk of cancer; in Cockayne, the repair defect can cause developmental and neurological disease.

While the idea of transcribing and repairing DNA at the same time is nothing new, Nudler’s team has specified the mechanism of such transcription-coupled repair that allows RNA polymerase help maintain the integrity of the E. coli genome. “Whatever the DNA damage, RNA polymerase will probably be the first to recognize it,” said Nudler.

Moreover, Nudler suggested his team’s work could help explain the puzzling abundance of RNAs observed in human cells. In the last few years, researchers have recognized that many more RNA molecules are transcribed than are translated into proteins. “The boundaries between genic and nongenic regions are becoming more blurry,” said Thomas Gingeras, a biologist at Cold Spring Harbor Laboratory in New York.

And it appears RNA polymerase is working overtime: a handful of studies published in the last decade have suggested that the genome is “pervasively transcribed,” with up to 76 percent of the DNA converted into RNA. The question, then, as Nudler put it, is: “Why do these regions need to be transcribed? Why waste your energy to synthesize so many RNA transcripts?”

V. Epshtein et al., “UvrD facilitates DNA repair by pulling RNA polymerase backwards,” Nature, doi:10.1038/nature12928, 2014.


see more:
http://reasonandscience.heavenforum.org/t2043-nucleotide-excision-repair#3475

1) http://en.wikipedia.org/wiki/Transcription_factor_II_H
2) http://www.biochem.umd.edu/biochem/kahn/molmachines/newpolII/TFIIH.html
3) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC23002/
4) http://www.genome.jp/dbget-bin/www_bget?ko03420
5) https://en.wikipedia.org/wiki/Nucleotide_excision_repair#Transcription_coupled_repair_.28TC-NER.29
6) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4049952/figure/F1/
7) http://www.the-scientist.com/?articles.view/articleNo/39137/title/DNA-Damage-Scout/



Last edited by Admin on Sat Jun 20, 2015 7:36 am; edited 16 times in total

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Transcription elongation factor TFIIS

TFIIS is a component of RNA polymerase II preinitiation complexes, and is required for preinitiation complex assembly and stability 1

TFII S is an elongation factor that is responsible to stimulate elongation but not initiation transcription (Weaver, 2002).  One primary function of TFII S is that it allows the RNA polymerase to transcribe through the pause sites of DNA (Pan et al, 1997 and Weaver, 2002).  A pause site is a site where RNA polymerase pauses at before it continues to transcribe again (Weaver, 2002).  The pause sites are overcome by the binding of TFII S to RNA polyermase II, followed by the cleaving of the 3’ end of the nascent RNA.  After the cleavage of the nascent RNA chain, the RNA polymerase then reacts with the new 3’ end, causing the minimization of the pause sites (Pan et al, 1997).
Besides from helping the RNA polymerase to overcome the pause sites, TFII S can also proofread and induce the enzyme of RNA polymerase to remove any mistaken nucleotides (Weaver, 2002).    


It was found that eukaryotic TFII S showed a great deal of homologies among themselves.  However, the eukaryotic TFII S does not work with the bacterial RNA polymerase or vice versa (Shimasaki et al 2000).  TFII S consistsed of three domains:  Domain I, Domain II and Domain III.  Domain I is the least conserved among the mammalian species (Shimasaki et al 2000).  Domain II is an alpha – helical region in which it is a site where the binding of RNA polymerase occurs (Shimasaki et al 2000).  Domain III is the most conserved region among the three domains (Shimasaki et al 2000).  Domain III has a “zinc ribbon motif” that is comparable to some nuclear polymerases’ subunits (Shimasaki et al 2000). 2







1) http://www.ebi.ac.uk/interpro/entry/IPR006289
2) http://www.biochem.umd.edu/biochem/kahn/molmachines/newpolII/TFIIS.html

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DNA repair mechanisms, designed with special care in order to provide integrity of DNA, and  essential for living organisms of all domains.

Maintaining the genetic stability that an organism needs for its survival requires not only an extremely accurate mechanism for replicating DNA, but also mechanisms for repairing the many accidental lesions that occur continually in DNA.

DNA damage is an alteration in the chemical structure of DNA, such as a break in a strand of DNA, a base missing from the backbone of DNA, or a chemically changed base.  Naturally occurring DNA damages arise more than 60,000 times per day per mammalian cell.   DNA damage appears to be a fundamental problem for life. DNA damages are a major primary cause of cancer. DNA damages give rise to mutations and epimutations. The mutations, if not corrected,  would be propagated throughout subsequent cell generations. Such a high rate of random changes in the DNA sequence would have disastrous consequences for an organism

Different pathways for DNA repair exists, Nucleotide excision repair (NER),  Base excision repair (BER),  DNA mismatch repair (MMR),  Repair through alkyltransferase-like proteins (ATLs) amongst others.

Its evident that the repair mechanism is essential for the cell to survive. It could not have evolved after life arose, but must have come into existence before. The mechanism is highly complex and elaborated, as consequence, the design inference is justified and seems to be the best way to explain its existence.

Base excision repair (BER)  involves a category of enzymes  known as  DNA-N-glycosylases.

One example of DNA's  automatic error-correction utilities are enough to stagger the imagination.  There are dozens of repair mechanisms to shield our genetic code from damage; one of them was portrayed in Nature  in terms that should inspire awe.

From Nature's article :
Structure of a repair enzyme interrogating undamaged DNA elucidates recognition of damaged DNA 11

How DNA repair proteins distinguish between the rare sites of damage and the vast expanse of normal DNA is poorly understood. Recognizing the mutagenic lesion 8-oxoguanine (oxoG) represents an especially formidable challenge, because this oxidized nucleobase differs by only two atoms from its normal counterpart, guanine (G).  The X-ray structure of the trapped complex features a target G nucleobase extruded from the DNA helix but denied insertion into the lesion recognition pocket of the enzyme. Free energy difference calculations show that both attractive and repulsive interactions have an important role in the preferential binding of oxoG compared with G to the active site. The structure reveals a remarkably effective gate-keeping strategy for lesion discrimination and suggests a mechanism for oxoG insertion into the hOGG1 active site.

Of the four bases in DNA (C, G, A, and T) cytosine or C is always supposed to pair with guanine, G, and adenine, A, is always supposed to pair with thymine, T.  The enzyme studied by Banerjee et al. in Nature is one of a host of molecular machines called BER glycosylases; this one is called human oxoG glycosylase repair enzyme (hOGG1), and it is specialized for finding a particular type of error: an oxidized G base (guanine).  Oxidation damage can be caused by exposure to ionizing radiation (like sunburn) or free radicals roaming around in the cell nucleus.  The normal G becomes oxoG, making it very slightly out of shape.  There might be one in a million of these on a DNA strand.  While it seems like a minor typo, it can actually cause the translation machinery to insert the wrong amino acid into a protein, with disastrous results, such as colorectal cancer.  12

The machine latches onto the DNA double helix and works its way down the strand, feeling every base on the way.  As it proceeds, it kinks the DNA strand into a sharp angle.  It is built to ignore the T and A bases, but whenever it feels a C, it knows there is supposed to be a G attached.  The machine has precision contact points for C and G.  When the C engages, the base paired to it is flipped up out of the helix into a slot inside the enzyme that is finely crafted to mate with a pure, clean G.  If all is well, it flips the G back into the DNA helix and moves on.  If the base is an oxoG, however, that base gets flipped into another slot further inside, where powerful forces yank the errant base out of the strand so that other machines can insert the correct one.

Now this is all wonderful stuff so far, but as with many things in living cells, the true wonder is in the details.  The thermodynamic energy differences between G and oxoG are extremely slight – oxoG contains only one extra atom of oxygen – and yet this machine is able to discriminate between them to high levels of accuracy.

The author, David, says in the Nature article :

Structural biology:  DNA search and rescue

DNA-repair enzymes amaze us with their ability to search through vast tracts of DNA to find subtle anomalies in the structure. The human repair enzyme 8-oxoguanine glycosylase (hOGG1) is particularly impressive in this regard because it efficiently removes 8-oxoguanine (oxoG), a damaged guanine (G) base containing an extra oxygen atom, and ignores undamaged bases.

Natural selection cannot act without accurate replication, yet the protein machinery for the level of accuracy required is itself built by the very genetic code it is designed to protect.  Thats a catch22 situation.  It would have been challenging enough to explain accurate transcription and translation alone by natural means, but as consequence of UV radiation, it  would have quickly been destroyed through accumulation of errors.  So accurate replication and proofreading are required for the origin of life. How on earth could proofreading enzymes emerge, especially with this degree of fidelity, when they depend on the very information that they are designed to protect?  Think about it....  This is one more prima facie example of chicken and egg situation. What is the alternative explanation to design ? Proofreading DNA by chance ?  And a complex suite of translation machinery without a designer?  

I  enjoy to learn about  the wonder of these incredible mechanisms.  If the apostle Paul could understand that creation demands a Creator as he wrote in Romans chapter one 18, how much more we today with all the revelations about cell biology and molecular machines?  

http://reasonandscience.heavenforum.org/t2043-dna-repair#3475

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How 'molecular machines' inside cells swing into action to activate genes at different times in a cell's life is revealed today in new research published in Molecular Cell.

http://www3.imperial.ac.uk/newsandeventspggrp/imperialcollege/newssummary/news_7-11-2008-12-19-59

http://m.phys.org/news/2008-11-molecular-machines-gene-revealed.html

How 'molecular machines' inside cells swing into action to activate genes at different times in a cell's life is revealed today in new research published in Molecular Cell.
Genes are made of double stranded DNA molecules containing the coded information an organism's cells need to produce proteins. The DNA double strands need to be 'melted out' and separated in order for the code to be accessed. Once accessed, the genetic codes are converted to messenger RNAs (mRNA) which are used to make proteins. Cells need to produce particular proteins at different times in their lives, to help them respond and adapt to changes in their environment.

The new study outlines exactly how a molecular machine called RNA polymerase, which reads the DNA code and synthesizes mRNA, is kickstarted by specialised activator proteins. The scientists have discovered that RNA polymerase uses a tightly regulated internal blocking system that prevents genes from being activated when they are not needed.

Using electron microscopy to look at the inner workings of bacterial cells, the researchers discovered that the DNA strand-separating process is kickstarted when RNA polymerase is modified by an activator protein, which the cell sends to the site of the gene that needs to be switched on.

This activator protein jump-starts the RNA polymerase machine by removing a plug which blocks the DNA's entrance to the machine. The activator protein also causes the DNA strands to shift position so that the DNA lines up with the entrance to the RNA polymerase. Once these two movements have occurred and the DNA strands are in position, the RNA polymerase machine gets to work melting them out, so that the information they contain can be processed to produce mRNA, and ultimately allow production of proteins.

Professor Xiaodong Zhang, lead author of the paper from the Department of Life Sciences at Imperial College London, explains the significance of the team's findings, saying:

"Understanding how the RNA polymerase gene transcription 'machine' is activated, and how it is stalled from working when it is not needed, gives us a better insight than ever before into the inner workings of cells, and the complex processes that occur to facilitate the carefully regulated production of proteins."



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