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The RNA polymerase enzyme and its function, evidence of design

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The RNA polymerase enzyme and its function, evidence of design

http://reasonandscience.heavenforum.org/t2016-the-rna-polymerase-enzyme-and-its-function-evidence-of-design

DNA, the double-stranded molecule that carries genetic information and makes up chromosomes, transcribes when mRNA makes copy of a DNA strand. In this way, cells make proteins that help the cell do any number of things, they make proteins that are essential to the life of the organism. This process begins with an enzyme called RNA polymerase.  It's this enzyme that splits the DNA strands in two: a template strand and a coding strand. RNA uses that coding strand to transcribe information in the DNA to make a variety of "gene products" supporting the organism-including more RNA polymerase. A complex system of molecules is doing very specific things in very specific places at very specific times at very specific rates.It's a complex arrangement of six chains of different amino acids—over a thousand per chain. If you change the structure or remove even a few of the amino acids, he says, the enzyme's function collapses.

To describe the genesis, divergence and complexity of life on Earth, one must understand two major transitions: 1) between the ancient RNA-protein world and LUCA (the last universal common ancestor); and 2) between LUCA and LECA (the last eukaryotic common ancestor). On Earth, life divides into three domains: bacteria, archaea and eukaryotes.

Information flow within living organisms requires genetic material in DNA to be read and deciphered. Because, within the double helix, DNA bases project inward, DNA is more stable than single-stranded RNA, in which bases are more exposed. LUCA, therefore, developed a more stable DNA genome than was possible in the RNA-protein world. The DNA double helix, however, is difficult to unzip, so proteins must act on DNA to separate strands. The relative security of a DNA genome compared to a fragmented RNA genome comes with conditions.  

LUCA (~3.5 to 3.8 billion years ago) was one of the first organisms with a streamlined DNA genome. The Earth is only ~4.5 billion years old, so LUCA is very ancient and the time for the RNA-protein world that preceded LUCA was short. To read the genetic information within a DNA genome requires a mobile protein factory called RNA polymerase. To initiate RNA synthesis on a DNA template, RNA polymerases found in the three domains of life require protein factors that help them to recognize and open a promoter DNA sequence. In bacteria, sigma factors help RNA polymerases bind promoter DNA.

The evolutionary relationship, among bacterial sigma factors and archaeal TFB-TBP has not clearly been known.

Burton and Burton hypothesize that both sigma factors and TFB arose from a primordial initiation factor at LUCA with 4-HTH motifs, and bacteria and archaea diverged in evolution about the time of LUCA largely because of this difference in genome reading styles. Archaeal TFB lost 2-HTH motifs and gained compensating functions including cooperation with TBP. Because bacteria and archaea interpret (read; "transcribe") their DNA genomes differently to synthesize RNA, these organisms became distinct.

Question: how and why should that distinction have evolved ? the differences of transcriptions are hudge, eukaryotic cells use over 100 protein sub units.

Evolution of promoter DNA sequences has long been a mystery, but bacterial promoters somewhat resemble archaeal and eukaryotic promoters.

Its still today a mystery, and to the well intended its obvious why. The resemblance of one with the other means nothing.

very early in evolution, bacteria and archaea came to read and interpret genomic information in DNA in fundamentally different ways, causing bacteria and archaea to diverge into distinct domains.

Question: how did they " learn " that feat ??

At LECA (~1.6-2.5 billion years ago) an archaea fused with multiple bacteria to form the first eukaryote.

Cool story. How do they possibly know ? Answer : they don't. There are hudge differences between the two :

Gene expression is linked to RNA transcription, which cannot happen without RNA polymerase. However, this is where the similarities between prokaryote and eukaryote expression end.

Transcription in Bacteria
In bacteria, all transcription is performed by a single type of RNA polymerase. This polymerase contains four catalytic subunits and a single regulatory subunit known as sigma (s). Interestingly, several distinct sigma factors have been identified, and each of these oversees transcription of a unique set of genes. Sigma factors are thus discriminatory, as each binds a distinct set of promoter sequences.

Transcription in Eukaryotes
Eukaryotic cells are more complex than bacteria in many ways, including in terms of transcription.
Specifically, in eukaryotes, transcription is achieved by three different types of RNA polymerase

These polymerases differ in the number and type of subunits they contain, as well as the class of RNAs they transcribe; that is, RNA pol I transcribes ribosomal RNAs (rRNAs), RNA pol II transcribes RNAs that will become messenger RNAs (mRNAs) and also small regulatory RNAs, and RNA pol III transcribes small RNAs such as transfer RNAs (tRNAs). Because RNA pol II transcribes protein-encoding genes, it has been of particular importance to scientists who study the regulation of eukaryotic gene expression, and its function is well understood. For example, researchers know that RNA pol II can bind to a DNA sequence within the promoter of many genes, known as the TATA box, to initiate transcription.

So there are remarkable differences and a dramatic increase of complexity of this machinery between prokaryotic and eukaryotic cells. Its hard to see how and why there could have been the evolution from one to the other 3 in eukaryotic cells.

Together with other common motifs (short recognition sequences in the DNA), these elements constitute the core promoter.

Bacterial RNA polymerase is a huge factory with many moving parts. It is composed of a dozen different proteins. Together, they form a machine that surrounds DNA strands, unwinds them, and builds an RNA strand based on the information held inside the DNA. Once the enzyme gets started, RNA polymerase marches confidently along the DNA copying RNA strands thousands of nucleotides long.  

Observe the nomenclature : huge factory, machine, marches. Question : how did the dozens of proteins arise ? Unless this protein machine was not fully formed, no mRNA could be formed, no transcription and subsequently, no replication would be possible, upon which mutations and natural selection could act. So the only alternatives left are chance, physical necessity, and design. Could it be that chance, or physical necessity are good explanations for the arise of the enormously complex RNA polymerase machine?

Accuracy

As you might expect, RNA polymerase needs to be accurate in its copying of genetic information. To improve its accuracy, it performs a simple proofreading stepas it builds an RNA strand.

Question: unless enough transcription accuracy is achieved, the whole process would not be accurate enough, and the whole process would be compromised. How could it have got this amazing feature upon natural ways ? Isnt that far better explained through a intentional designer, who created this essential mechanism ?

The active site is designed to be able to remove nucleotides as well as add them to the growing strand. The enzyme tends to hover around mismatched nucleotides longer than properly added ones, giving the enzyme time to remove them.

Question : how did the machine arise this amazing ability ?

This process is somewhat wasteful, since proper nucleotides are also occasionally removed, but this is a small price to pay for creating better RNA transcripts. Overall, RNA polymerase makes an error about once in 10,000 nucleotides added, or about once per RNA strand created.

Question: How did it get such a amazing accuracy ?

To transcribe a gene accurately, RNA polymerase must recognize where on the genome to start and where to finish.

Question: Had RNA polymerase not to be able to recognize where to start right from the beginning, otherwise it would not be functional ? How did it learn that feat ?  

The initiation of transcription is an especially important step in gene expression because it is the main point at which the cell regulates which proteins are to be produced and at what rate.

Question : had the cell not to be able right from the start of its operation to regulate which proteins had to be produced, and and what rate ? How did it " learn " that feat ? Unless it was able to do so right from the beginning, would chaos not be the result ? Would the cell not cease to function properly ?

The bacterial RNA polymerase core enzyme is a multisubunit complex that sythesizes RNA using a DNA template as a guide.

Question : Unless it uses a DNA template as a guide, would the whole process also not be functional ? Had it not have to be able to use the DNA template as a guide right from the start ?

A detachable subunit called sigma (s) factor associates with the core enzyme and assists it in reading the signals in the DNA that tell it where to begin transcribingThese proteins belong to a family of polypeptides and they have very interesting features and play important role in specific gene expression.
They associate with RNAP subunits and they are also responsible for identifying the correct promoter and positioning the RNAP on DNA in proper context for initiating transcription. Otherwise the RNAP without sig cannot function as an enzyme; it is like a driver without a rudder/conductor.

Sigma (s) is a dissociable subunit of RNAP. When s is bound to core, the resulting complex, called holoenzyme, can bind with high affinity to promoter sites.

Without the sigma factor, RNA polymerase would bind loosely and randomly to DNA, but would not be able to start transcription.  Therefore no proteins would be made and the cell would die. 4)

Question: the sigma factor alone is LIFE ESSENTIAL. Had it not have to be there fully functional and programmed to do its job right when life started ?

It seems the RNA polymerase enzyme could not be a product of  chance,
necessity (or natural law), or a  combination of chance and necessity, and neither evolution, since evolution only works after replication is in place. The best probabilistic explanation is therefore design. Is this a argument from ignorance ? I don't think so.

http://www.arn.org/docs/booher/scientific-case-for-ID.html

This objection is essentially stating that intelligent design is just a mysterious notion that covers for ignorance—a sort of God of the gaps argument. But the design inference does not constitute an argument from ignorance, rather it constitutes an “inference to the best explanation” based on our best available knowledge. “We are not ignorant of how information arises. We know from experience that conscious intelligent agents can create informational sequences and systems…. When we encounter the information based in large biological molecules needed for life, we can infer—based on our knowledge of established cause and effect relationship—that an intelligent cause operated in the past to produce the specified information necessary to the origin of life.” Intelligent design is applying a standard historical sciences principle of uniformitarianism, that “the present is the key to the past.”




DNA, the double-stranded molecule that carries genetic information and makes up chromosomes, transcribes when mRNA makes copy of a DNA strand. In this way, cells make proteins that help the cell do any number of things, they make proteins that are essential to the life of the organism. This process begins with an enzyme called RNA polymerase.  It's this enzyme that splits the DNA strands in two: a template strand and a coding strand. RNA uses that coding strand to transcribe information in the DNA to make a variety of "gene products" supporting the organism-including more RNA polymerase. A complex system of molecules is doing very specific things in very specific places at very specific times at very specific rates.It's a complex arrangement of six chains of different amino acids—over a thousand per chain. If you change the structure or remove even a few of the amino acids, he says, the enzyme's function collapses  11

To describe the genesis, divergence and complexity of life on Earth, one must understand two major transitions: 1) between the ancient RNA-protein world and LUCA (the last universal common ancestor); and 2) between LUCA and LECA (the last eukaryotic common ancestor). On Earth, life divides into three domains: bacteria, archaea and eukaryotes. 7

Information flow within living organisms requires genetic material in DNA to be read and deciphered. Because, within the double helix, DNA bases project inward, DNA is more stable than single-stranded RNA, in which bases are more exposed. LUCA, therefore, developed a more stable DNA genome than was possible in the RNA-protein world. The DNA double helix, however, is difficult to unzip, so proteins must act on DNA to separate strands. The relative security of a DNA genome compared to a fragmented RNA genome comes with conditions.  

LUCA (~3.5 to 3.8 billion years ago) was one of the first organisms with a streamlined DNA genome. The Earth is only ~4.5 billion years old, so LUCA is very ancient and the time for the RNA-protein world that preceded LUCA was short. To read the genetic information within a DNA genome requires a mobile protein factory called RNA polymerase. To initiate RNA synthesis on a DNA template, RNA polymerases found in the three domains of life require protein factors that help them to recognize and open a promoter DNA sequence. In bacteria, sigma factors help RNA polymerases bind promoter DNA.

The evolutionary relationship, among bacterial sigma factors and archaeal TFB-TBP has not clearly been known.

Burton and Burton hypothesize that both sigma factors and TFB arose from a primordial initiation factor at LUCA with 4-HTH motifs, and bacteria and archaea diverged in evolution about the time of LUCA largely because of this difference in genome reading styles. Archaeal TFB lost 2-HTH motifs and gained compensating functions including cooperation with TBP. Because bacteria and archaea interpret (read; "transcribe") their DNA genomes differently to synthesize RNA, these organisms became distinct.

Question: how and why should that distinction have evolved ? the differences of transcriptions are hudge, eukaryotic cells use over 100 protein sub units.

Evolution of promoter DNA sequences has long been a mystery, but bacterial promoters somewhat resemble archaeal and eukaryotic promoters.

Its still today a mystery, and to the well intended its obvious why. The resemblance of one with the other means nothing.

very early in evolution, bacteria and archaea came to read and interpret genomic information in DNA in fundamentally different ways, causing bacteria and archaea to diverge into distinct domains.

Question: how did they " learn " that feat ??  

At LECA (~1.6-2.5 billion years ago) an archaea fused with multiple bacteria to form the first eukaryote.

Cool story. How do they possibly know ? Answer : they don't. There are hudge differences between the two :


Gene expression is linked to RNA transcription, which cannot happen without RNA polymerase. However, this is where the similarities between prokaryote and eukaryote expression end. 8

Transcription in Bacteria
In bacteria, all transcription is performed by a single type of RNA polymerase. This polymerase contains four catalytic subunits and a single regulatory subunit known as sigma (s). Interestingly, several distinct sigma factors have been identified, and each of these oversees transcription of a unique set of genes. Sigma factors are thus discriminatory, as each binds a distinct set of promoter sequences.

Transcription in Eukaryotes
Eukaryotic cells are more complex than bacteria in many ways, including in terms of transcription.
Specifically, in eukaryotes, transcription is achieved by three different types of RNA polymerase

These polymerases differ in the number and type of subunits they contain, as well as the class of RNAs they transcribe; that is, RNA pol I transcribes ribosomal RNAs (rRNAs), RNA pol II transcribes RNAs that will become messenger RNAs (mRNAs) and also small regulatory RNAs, and RNA pol III transcribes small RNAs such as transfer RNAs (tRNAs). Because RNA pol II transcribes protein-encoding genes, it has been of particular importance to scientists who study the regulation of eukaryotic gene expression, and its function is well understood. For example, researchers know that RNA pol II can bind to a DNA sequence within the promoter of many genes, known as the TATA box, to initiate transcription.

So there are remarkable differences and a dramatic increase of complexity of this machinery between prokaryotic and eukaryotic cells. Its hard to see how and why there could have been the evolution from one to the other 3 in eukaryotic cells.

Together with other common motifs (short recognition sequences in the DNA), these elements constitute the core promoter.

So there is not only a difference between the RNA polymerase machinery, but also in the promoters sections in DNA.

Prokaryotic vs. Eukaryotic Trancription
Differences:
Prokaryotes only contain three different promoter elements: -10, -35 promoters, and upstream elements.  Eukaryotes contain many different promoter elements: TATA box, initiator elements, downstream core promoter element, CAAT box, and the GC box to name a few.  Eukaryotes have three types of RNA polymerases, I, II, and III, and prokaryotes only have one type.  Eukaryotes form and initiation complex with the various transcription factors that dissociate after initiation is completed.  There is no such structure seen in prokaryotes.  Another main difference between the two is that transcription and translation occurs simultaneously in prokaryotes and in eukaryotes the RNA is first transcribed in the nucleus and then translated in the cytoplasm.  RNAs from eukaryotes undergo post-transcriptional modifications including: capping, polyadenylation, and splicing.  These events do not occur in prokaryotes.  mRNAs in prokaryotes tend to contain many different genes on a single mRNA meaning they are polycystronic.  Eukaryotes contain mRNAs that are monocystronic.  Termination in prokaryotes is done by either rho-dependent or rho-independent mechanisms.  In eukaryotes transcription is terminated by two elements: a poly(A) signal and a downstream terminator sequence. 9

These are hudge differences. How could or should the transition from one kind of promoters and RNA polymerase machinery have occured to the other domain, far more complex ?

The RNA in a cell is made by DNA transcription, a process that has certain similarities to the process of DNA replication. The machines doing this job, RNA polymerase enzymes,  are essential to life and are found in all organisms and many viruses.

By selecting which RNAs are made, RNA polymerase (RNAP) dictates how cells adapt to new environments, interact symbiotically or pathogenically with hosts, respond to stress and starvation, and multiply. RNAP accomplishes this task by deciding where and how often to start transcription, how to elongate RNAs, and where to stop. 6

Question: How did the cell " learn " how to decide where, and how often to start transcription ? Had this mechanism not have to be in place before the first cell was ready to replicate ?

Emergence of transcription initiation factors in the three domains of life.
The bacterial RNA polymerase (RNAP) strictly depends on σ-factors for transcription initiation, whereas archaeal and eukaryotic RNAPs require the initiation factors TATA box-binding protein (TBP) and transcription factor IIB (TFIIB). There are four potential scenarios for the evolution of these factors. As the first scenario requires the fewest independent evolutionary events, it is the simplest explanation for the occurrence of transcription initiation factors in all extant life. a | As there are no σ-factor homologues in archaea and eukaryotes, and no TBP and TFIIB homologues in bacteria, it is likely that the ancestral RNAP of the last universal common ancestor (LUCA) used neither σ-factors nor TBP or TFIIB factors, and that these factors evolved independently in the bacterial and archaeal–eukaryotic lineages, respectively, after their split. b | An alternative scenario is that the RNAP of the LUCA used both σ-factors and TBP and TFIIB factors in parallel, and then lost the relevant factors in each lineage. c | A third scenario is that the LUCA used σ-factors, and then the archaeal–eukaryotic lineage lost these factors are gained TBP and TFIIB factors. d | The final scenario is that the LUCA used TBP and TFIIB factors, and that these were then lost in the bacterial lineage and σ-factors were gained.10



There are four potential scenarios for the evolution of these transcription factors. Translated : they have no idea how the different factors could have evolved , and the mechanisms involved in a detailled manner. They just make things up. If someone expected to read the paper on how this large, complex transcription machine evolved, you would be disappointed.  The authors merely assume it evolved, and attempt to place various scenarios into an evolutionary tree where they don’t fit very well.  This is typical of evolutionary scientific literature that already “knows” evolution is a “fact” but has no way to back up the claim. Highly-complex, interdependent systems that involves a coded , specified, complex language,  translation, regulation, feedback, quality control and highest precision. The paper above fails completely to address the origin of the most efficient information-processing system known on earth. And so do all mainstream scientific papers. Their frequent answer is : " We don't know yet ". As if one day they would know, and evolution would be confirmed...... Wait a minute. Really ??  

Bacterial RNA polymerase is a huge factory with many moving parts. It is composed of a dozen different proteins. Together, they form a machine that surrounds DNA strands, unwinds them, and builds an RNA strand based on the information held inside the DNA. Once the enzyme gets started, RNA polymerase marches confidently along the DNA copying RNA strands thousands of nucleotides long.  1

Observe the nomenclature : huge factory, machine, marches. Question : how did the dozens of proteins arise ? Unless this protein machine was not fully formed, no mRNA could be formed, no transcription and subsequently, no replication would be possible, upon which mutations and natural selection could act. So the only alternatives left are chance, physical necessity, and design. Could it be that chance, or physical necessity are good explanations for the arise of the enormously complex RNA polymerase machine?

Accuracy

As you might expect, RNA polymerase needs to be accurate in its copying of genetic information. To improve its accuracy, it performs a simple proofreading stepas it builds an RNA strand.

Question: unless enough transcription accuracy is achieved, the whole process would not be accurate enough, and the whole process would be compromised. How could it have got this amazing feature upon natural ways ? Isnt that far better explained through a intentional designer, who created this essential mechanism ?

The active site is designed to be able to remove nucleotides as well as add them to the growing strand. The enzyme tends to hover around mismatched nucleotides longer than properly added ones, giving the enzyme time to remove them.

Question : how did the machine arise this amazing ability ?

This process is somewhat wasteful, since proper nucleotides are also occasionally removed, but this is a small price to pay for creating better RNA transcripts. Overall, RNA polymerase makes an error about once in 10,000 nucleotides added, or about once per RNA strand created.

Question: How did it get such a amazing accuracy ?

To transcribe a gene accurately, RNA polymerase must recognize where on the genome to start and where to finish.

Question: Had RNA polymerase not to be able to recognize where to start right from the beginning, otherwise it would not be functional ? How did it learn that feat ?  

The initiation of transcription is an especially important step in gene expression because it is the main point at which the cell regulates which proteins are to be produced and at what rate.

Question : had the cell not to be able right from the start of its operation to regulate which proteins had to be produced, and and what rate ? How did it " learn " that feat ? Unless it was able to do so right from the beginning, would chaos not be the result ? Would the cell not cease to function properly ?

The bacterial RNA polymerase core enzyme is a multisubunit complex that sythesizes RNA using a DNA template as a guide.

Question : Unless it uses a DNA template as a guide, would the whole process also not be functional ? Had it not have to be able to use the DNA template as a guide right from the start ?

A detachable subunit called sigma (s) factor associates with the core enzyme and assists it in reading the signals in the DNA that tell it where to begin transcribing

Sigma factors:

http://en.wikipedia.org/wiki/Sigma_factor





These proteins belong to a family of polypeptides and they have very interesting features and play important role in specific gene expression.
They associate with RNAP subunits and they are also responsible for identifying the correct promoter and positioning the RNAP on DNA in proper context for initiating transcription. Otherwise the RNAP without sig cannot function as an enzyme; it is like a driver without a rudder/conductor.

Sigma (s) is a dissociable subunit of RNAP. When s is bound to core, the resulting complex, called holoenzyme, can bind with high affinity to promoter sites. 3)

Without the sigma factor, RNA polymerase would bind loosely and randomly to DNA, but would not be able to start transcription.  Therefore no proteins would be made and the cell would die. 4)

Question: the sigma factor alone is LIFE ESSENTIAL. Had it not have to be there fully functional and programmed to do its job right when life started ?


Core RNA polymerase in bacteria is a complex composed of an

two alpha subunits
one beta, one beta’
one omega subunit,

together they form the core structure.

The α-amino-terminal domains (α-NTDs) serve as a scaffold for complex assembly;
the α-carboxy-terminal domains (α-CTDs) and ω-subunit play regulatory roles during initiation.
The β- and β′-subunits jointly form the active site and make all the contacts to the nucleic acids.






image ru



The process of transcription can be divided into three stages:

1) Initiation of a new RNA chain  (parts 1-4 of the picture below).
2) Elongation of the chain  (parts 5&6 of the picture below).
3) Termination of transcription and release of the brand new RNA molecule  (parts 6&7 of the picture below).


upload pic

Together, s factor and core enzyme are known as the RNA polymerase holoenzyme; this complex adheres only weakly to bacterial DNA when the two collide, and a holoenzyme typically slides rapidly along the long DNA molecule until it dissociates again. However, when the polymerase holoenzyme slides into a region on the DNA double helix called a promoter, a special sequence of nucleotides indicating the starting point for RNA synthesis, the polymerase binds tightly to this DNA.

Question: Is it not essential, that this special sequence of nucleotides is there in the DNA code right from the beginning, otherwise the right mRNA cannot be synthesized ? How did these special sequences get there naturally ? How did the polymerase " learn " to bind tightly to this DNA ? Had it not have to be able to bind tightly right from the beginning ? Are these not functions, that are essential, and if they are not fully functional right from the beginning, they do not function at all ?  

The polymerase holoenzyme, through its s factor, recognizes the promoter DNA sequence by making specific contacts with the portions of the bases that are exposed on the outside of the helix step 1 in the  Figure above.  After the RNA polymerase holoenzyme binds tightly to the promoter DNA in this way, it opens up the double helix to expose a short stretch of nucleotides on each strand (step 2 )

Question: how did the holoenzyme " learn " to do this precise step in a functional and correct way ? Try and fail attempts would not permit the process to go ahead, and the whole process would be compromised. Had the holoenzyme not have to be functional and fully formed right from the beginning ?

With the DNA unwound, one of the two exposed DNA strands acts as a template for complementary base-pairing with incoming ribonucleotides, two of which are joined together by the polymerase to begin an RNA chain (step 3 )



Questions:

Had the incoming and exit movement of the DNA strand not have to be pre programmed  ?
Had the enter and exit channel of DNA not have to be precisely designed, and be there right from the beginning ? There could be no function of a incompletely designed enzyme.
Had the incoming and exit movement of the ribonucleotides and newly formed mRNA strand not have to be pre programmed  ?
Had the enter and exit channel of ribonucleotides and the mRNA strand  not have to be precisely designed, and be there right from the beginning ?

Had the ribonucleotides not have to be readily availabe for recruitment right from the beginning ? Had the entry channel and the exit channel of  the ribonucleotides to enter and leave  the factory and being moved and released to the place where the action goes on not to be designed?  


After the first ten or so nucleotides of RNA have been synthesized (a relatively inefficient process during which polymerase synthesizes and discards short RNA oligomers), the core enzyme breaks its interactions with the promoter DNA, weakens its interactions with s factor, and begins to move down the DNA, synthesizing RNA (steps 4 and 5 )

Chain elongation continues (at a speed of approximately 50 nucleotides/sec for bacterial RNA polymerases) until the enzyme encounters a second signal in the DNA, the terminator , where the polymerase halts and releases both the newly made RNA chain and the DNA template (step 7 ) After the polymerase core enzyme has been released at a terminator, it reassociates with a free s factor to form a holoenzyme that can begin the process of transcription again.

Termination signals induce rapid and irreversible dissociation of the nascent transcript from RNA polymerase. Terminators at the end of genes prevent unintended transcription into the downstream genes, whereas terminators in the upstream regulatory leader regions adjust expression of the structural genes in response to metabolic and environmental signals. Premature termination within an operon leads to potentially deleterious defects in the expression of the downstream genes, but also provides an important surveillance mechanism. 5

Question : had the terminator not have to be there right from the beginning, otherwise the new mRNA strand would not have the right end ?

The processes of transcription initiation and termination involve a complicated series of structural transitions in protein, DNA, and RNA molecules. Unless the the whole operational sequence of the transcription cycle  is not fully functional right from the start, transcription cannot occur. Take away just one of the steps, and the process ceases to function. Is it feasable and imaginable, that the RNA polymerase enzyme with its complex transcription process requiring many parts and subunits could be the product of random natural processes ?  Transcription is a irreducible process. Furthermore, without replication , there is no evolution. In order for replication to occur, the DNA polymerase and RNA polymerase machinery had to be fully operational right since the start of life, so the machinery had to exist prior mutations and natural selection could occur.

Core promotors have to be encoded in DNA, otherwise the RNA polymerase enzyme does not know where to start and end transcription. Without it, no life would exist. It is impossible that it got there by trial and error. Unless it was at the right place right from the start, life could not happen. Feel free to bring chance into the game or good luck. You are guillable to the extreme, doing so.   Either its fully functional, or it does not function at all. Is that not strong evidence for intelligent design ? If not, what explanation of origin fits better the observation described here ?  
[/justify]
http://reasonandscience.heavenforum.org/t2016-the-rna-polymerase-enzyme-and-its-function-evidence-of-design#3391

1) http://www.rcsb.org/pdb/101/motm.do?momID=40
2) http://www.proteopedia.org/wiki/index.php/2e2i
3) http://www.researchgate.net/profile/John_Helmann/publication/228031784_Sigma_Factors_in_Gene_Expression/links/0fcfd506ac99963842000000.pdf
4) http://helicase.pbworks.com/w/page/17605688/Matthew-Hunter
5) http://www.nature.com/nrmicro/journal/v9/n5/abs/nrmicro2560.html
6) http://landick.wisc.edu/images/geszvain_landick.pdf
7) https://bmb.natsci.msu.edu/about/directory/faculty/zachary-f-burton/genesis-of-life-on-earth/
8 )http://www.nature.com/scitable/topicpage/rna-transcription-by-rna-polymerase-prokaryotes-vs-961
9) http://www.chem.uwec.edu/webpapers2006/sites/demlba/folder/provseuk.html
10) http://www.nature.com/nrmicro/journal/v9/n2/fig_tab/nrmicro2507_F8.html
11) http://storage.cloversites.com/makinglifecountministriesinc/documents/Intelligent%20Design.pdf



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http://www-als.lbl.gov/index.php/holding/160--proofreading-rna-structure-of-rna-polymerase-iis-backtracked-state.html

the error rate is as low as one mistake for every 100,000 DNA base pairs transcribed—thanks in part to error correction by an RNA polymerase known as pol II, which "backtracks," or reverses, along the transcript to remove misincorporated or damaged nucleotides.

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Lets compare the DNA instruction code and cell machinery to the production process of a piston of a car engine. The manufacturing engineer's focus is to turn raw materials into a new piston in the most economic, efficient and effective way possible. The first step is composing drawings that visually communicate how the whole car motor, piston is to be constructed. Familiar symbols, perspectives, units of measurement etc. are used for easy understanding.Together, such conventions constitute a visual language, and help to ensure that the drawing is unambiguous and relatively easy to understand. These drafting conventions are condensed into commonly accepted standards and specifications that transcend the barrier of language making technical drawings a universal means of communicating complex mechanical concepts. In the layout, there we can find the global drawing of each part, and the implementation of the parts in the motor, its position , how its mounted etc. The piston has a part number, and upon that number, the employee can find the individual drawing of the piston with all measures, sizes, material composition etc, and the drawing can be copied, and the copy sent to the machinist at the factory, and the piston can be manufactured there. Unless the empolyee is informed of the parts number in the drawing of the global layout, he will not be able to find the individual drawing, and it will be impossible to make the pistion. Same in biological systems. Unless the RNA polymerase does not receive the instruction through the promoter in the DNA chain, it will be unable to find the right place to start the transcription process. So the promoter is essential. No promoter , no life. Unless it was there right from the start, no transcription, no protein, no life.


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4 3'-end Cleavage and Polyadenylation. on Mon May 18, 2015 8:00 am

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3'-end Cleavage and Polyadenylation.


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Genetic studies in yeast indicate that virtually every subunit of the core complex is essential – for viability and for pre-mRNA processing and polyadenylation in vitro and in vivo. 16  

Although polyadenylation is seen in almost all organisms, it is not universal. However, the wide distribution of this modification and the fact that it is present in organisms from all three domains of life implies that the last universal common ancestor of all living organisms, it is presumed, had some form of polyadenylation system 17

Introduction

Most of the RNA found in our cells is built using our DNA genome as a template. In special cases, however, our cells also build RNA strands without a template. For instance, the end of (almost) every messenger RNA strand is composed of a long string of repeated adenosine nucleotides. These long poly(A) tails are not encoded in the genome. Instead, they are added after RNA polymerase finishes its normal process of transcription.

Its evident that this process and the instructions in order for this to happen after RNA polymerase finished its job,  had to be pre-programmed in DNA.

After RNA polymerase releases the RNA strand, other enzymes add the finishing touches, editing out introns, adding a cap to the front end, and building the long poly(A) tail at the other end. 5

Add the finishing touches, editing, adding , building the long poly (A) tail at the other end are highly ordered and precise processes. They could not have arisen without a intelligence actually instructing these sequenced and ordered proceedings, just by natural random processes.  

The Tail End

A complex of over a dozen enzymes oversees the creation of a poly(A) tail on messenger RNA molecules.

Had these enzymes not have to be fully operational right from the start ? Unless they were fully working right from the start, the process could not happen.

Several special sequences at the end of the RNA recruit this complex to the proper place. Then the RNA strand is cleaved, and about 250 adenosine nucleotides are added to the new end. The enzyme poly(A) polymerase (PAP)  is responsible for the creation of the poly(A) tail. With the help of two magnesium ions, it binds to the messenger RNA and adds adenosine nucleotides one at a time to the end of the strand.

Recruit to the proper place, cleaving, adding...... are all organized precise processes like in a regular manufacturing process in factories. How did natural process find out that magnesium ions would be required for a binding process ??

Heads and Tails

The poly(A) tail plays several important roles in the function of messenger RNA molecules. With the help of poly(A)-binding protein , it protects the end of the RNA strand, shielding it from RNA-cutting nucleases. It also assists with the transport of the messenger RNA out of the nucleus through nuclear pores. Surprisingly, the poly(A) tail, which is at the end the messenger RNA, also stimulates the start of protein synthesis by helping to recruit translation initiation factors at the front end of the RNA. Some researchers actually think that poly(A)-binding protein links the RNA strand into a big circle. This could have a very useful consequence: since the beginning of the messenger RNA is so close to the end, ribosomes that have just finished making a protein could jump immediately to the beginning and start again.  5

12

Process of mRNA begins with transcription. Soon after RNA polymerase begins with transcription, a methylated cap is added to the 5" end. Transcription then ends to completion. Following completion, RNA polymerase releases the cap strand of mRNA. Specific nucleotide sequences in the mRNA are boung by  CstF: cleavage stimulation factor enzymes. The 3" end of the mRNA is then next moved into the right configuration for cleavage. Stabilizing factors are then added to the complex. Poly (A) polymerase now binds to the mRNA and cleaves the 3" end. The complex begins to disassociate, and the cleaved 3" end quickly degrades. Poly (A) polymerase now synthesizes the polyadenylated tail adenine to the tal. Additional proteins then add to the tail, increasing the rate at which it grows. When the tail reaches its full length, the poly (A) polymerase is signalled to stop , and the process is concluded. The processed mRNA is ready now to undergo splicing in preparation for translation.


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following enzyme proteins are involved in the process:

CPSF: cleavage/polyadenylation specificity factor
CstF: cleavage stimulation factor
PAP: polyadenylate polymerase
PABII: polyadenylate binding protein 2
CFI: cleavage factor I
CFII: cleavage factor II


that bind UGUA motifs upstream of the cleavage site12, 83 and two larger polypeptides. PAB, poly(A)-binding protein.  


The poly(A) tail consists of multiple adenosine monophosphates;

"Adenine synthesis is perhaps the best example of an irreducibly complex system that can be found in life ..."
the process doesn't work unless all 11 enzymes are present. 1

This process could not have happened for the first time prior the first self replicating cell arose. So evolution is excluded. Remains chance, physical necessity, or both together. Or design......

in other words, it is a stretch of RNA that has only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA (mRNA) for translation. It, therefore, forms part of the larger process of gene expression.

The process of polyadenylation begins as the transcription of a gene finishes, or terminates.

This process had to be instructed in order to begin and terminate at the right time. How was this instruction encoded into the genome ? What mechanism was involved ?

The 3'-most segment of the newly made pre-mRNA is first cleaved off by a set of proteins;

These proteins had to be in place already...... How was the instruction encoded to make them functional, and put them into the right place to operate?

these proteins then synthesize the poly(A) tail at the RNA's 3' end. In some genes, these proteins may add a poly(A) tail at any one of several possible sites. Therefore, polyadenylation can produce more than one transcript from a single gene (alternative polyadenylation), similar to alternative splicing.[1]
The poly(A) tail is important for the nuclear export, translation, and stability of mRNA.

So its basically another part that is essential for life. No poly (A) tail, no export, translation, and stability of mRNA.


Here, a multi-protein complex (see components on the right) cleaves the 3'-most part of a newly produced RNA and polyadenylates the end produced by this cleavage. The cleavage is catalysed by the enzyme CPSF

A complex machinery is involved in these reactions including the cleavage/polyadenylation specificity factor (CPSF) and the cleavage stimulation factor (CstF). A subunit of CPSF, CPSF-73, has been shown to be the enzyme responsible for the cleavage reaction (Dominski et al. 2005). 2
The rate at which PAP adds adenine nucleotides is dependent on the presence of another regulatory protein, PABPII (poly-adenine binding protein II).
The final tail is about 200-250 adenine nucleotides long. 3

So we need

cleavage/polyadenylation specificity factor(CPSF)


Cleavage and polyadenylation specificity factor (CPSF) is involved in the cleavage of the 3' signaling region from a newly synthesized pre-messenger RNA (pre-mRNA) molecule in the process of gene transcription. It is the first protein to bind to the signaling region near the cleavage site of the pre-mRNA, to which the poly(A) tail will be added by polynucleotide adenylyltransferase. The upstream signaling region has the canonical nucleotide sequence AAUAAA, which is highly conserved across the vast majority of pre-mRNAs. A second downstream signaling region, located on the portion of the pre-mRNA that is cleaved before polyadenylation, consists of a GU-rich region required for efficient processing.  13


Polynucleotide adenylyltransferase enzyme :

Atp + RNA(n) = diphosphate + RNA(n+1)

Question : What is the best explanation for the arise of this complex enzyme , its synthesis, and  its reaction   prior replication could occur ?


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cleavage stimulation factor (CstF)


Cleavage stimulatory factor or cleavage stimulation factor (CstF or CStF) is a heterotrimeric protein, made up of the proteins

CSTF1 (55kDa)
CSTF2 (64kDa)
CSTF3 (77kDa)


totalling about 200 kDa. It is involved in the cleavage of the 3' signaling region from a newly synthesized pre-messenger RNA (mRNA) molecule. CstF is recruited by cleavage and polyadenylation specificity factor (CPSF) and assembles into a protein complex on the 3' end to promote the synthesis of a functional polyadenine tail, which results in a mature mRNA molecule ready to be exported from the cell nucleus to the cytosol for translation. 4


CSTF1 (55kDa),

Cleavage stimulation factor 50 kDa subunit is a protein that in humans is encoded by the CSTF1 gene.
This gene encodes one of three subunits which combine to form cleavage stimulation factor (CSTF). CSTF is involved in the polyadenylation and 3'end cleavage of pre-mRNAs. Similar to mammalian G protein beta subunits, this protein contains transducin-like repeats. Several transcript variants with different 5' UTR, but encoding the same protein, have been found for this gene.  14 

CSTF2 (64kDa) and

Cleavage stimulation factor 64 kDa subunit is a protein that in humans is encoded by the CSTF2 gene.[1][2]
This gene encodes a nuclear protein with an RRM (RNA recognition motif) domain. The protein is a member of the cleavage stimulation factor (CSTF) complex that is involved in the 3' end cleavage and polyadenylation of pre-mRNAs. Specifically, this protein binds GU-rich elements within the 3'-untranslated region of mRNAs. 7


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CSTF3  (77kDa),

Cleavage stimulation factor 77 kDa subunit is a protein that in humans is encoded by the CSTF3 gene.
The protein encoded by this gene is one of three (including CSTF1 and CSTF2) cleavage stimulation factors that combine to form the cleavage stimulation factor complex (CSTF). This complex is involved in the polyadenylation and 3' end cleavage of pre-mRNAs. The encoded protein functions as a homodimer and interacts directly with both CSTF1 and CSTF2 in the CSTF complex. Alternative splicing results in multiple transcript variants encoding different isoforms. 8


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totalling about 200 kDa. It is involved in the cleavage of the 3' signaling region from a newly synthesized pre-messenger RNA (mRNA) molecule. CstF is recruited by cleavage and polyadenylation specificity factor (CPSF) and assembles into a protein complex on the 3' end to promote the synthesis of a functional polyadenine tail, which results in a mature mRNA molecule ready to be exported from the cell nucleus to the cytosol for translation.


A subunit of CPSF, CPSF-73

Schematic representation of the structure of human CPSF-73. The beta-strands and alpha-helices are labelled, and the two zinc atoms in the activesite are shown as grey spheres. The sulphate ion is shown as a stick model. 9

Our studies provide the first direct experimental evidence that CPSF-73 is the pre-mRNA 3′-end-processing endonuclease. 10 ( Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. )


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Polynucleotide adenylyltransferase (PAP)

This enzyme is responsible for the addition of the 3' polyadenine tail to a newly synthesized pre-messenger RNA (pre-mRNA) molecule during the process of gene transcription. The protein is the final addition to a large protein complex that also contains smaller assemblies known as the cleavage and polyadenylation specificity factor (CPSF) and cleavage stimulatory factor (CtSF) and its binding is a necessary prerequisite to the cleavage of the 3' end of the pre-mRNA. After cleavage of the 3' signaling region that directs the assembly of the complex, PAP adds the polyadenine tail to the new 3' end.
The rate at which PAP adds adenine nucleotides is dependent on the presence of another regulatory protein, PABPII (poly-adenine binding protein II). The first few nucleotides added by PAP are added very slowly, but the short polyadenine tail is then bound by PABPII, which accelerates the rate of adenine addition by PAP. The final tail is about 200-250 adenine nucleotides long. PAP is phosphorylated by mitosis-promoting factor , a key regulator of the cell cycle. High phosphorylation levels decrease PAP activity.


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PABPII (poly-adenine binding protein II)



1) http://reasonandscience.heavenforum.org/t2006-adenine-synthesis-in-life
2) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2151031/
3) http://en.wikipedia.org/wiki/Polynucleotide_adenylyltransferase
4) http://en.wikipedia.org/wiki/Cleavage_stimulation_factor
5) http://www.rcsb.org/pdb/101/motm.do?momID=106
6) http://www.nature.com/nrg/journal/v14/n7/fig_tab/nrg3482_F1.html
7) http://en.wikipedia.org/wiki/CSTF2
8 )http://en.wikipedia.org/wiki/CSTF3
9) http://www.nature.com/nature/journal/v444/n7121/fig_tab/nature05363_F1.html
10) http://www.nature.com/nature/journal/v444/n7121/full/nature05363.html#B2
11) http://en.wikipedia.org/wiki/CSTF1
12) https://www.youtube.com/watch?v=DoSRu15VtdM
13) http://en.wikipedia.org/wiki/Cleavage_and_polyadenylation_specificity_factor
14) http://en.wikipedia.org/wiki/Polynucleotide_adenylyltransferase
15) http://vcell.ndsu.nodak.edu/animations/mrnaprocessing/capping.htm
16) https://aghunt.wordpress.com/2008/08/08/giardia-lamblia-polyadenylation-and-irreducible-complexity/
17) http://en.wikipedia.org/wiki/Polyadenylation#Evolution

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5 RNA polymerase in eukaryotes on Tue Jun 02, 2015 10:39 pm

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RNA polymerase in eukaryotes

1,2

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

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 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.

RNA Polymerase II Requires General Transcription Factors

The general transcription factors help to position eucaryotic RNA polymerase correctly at the promoter.

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



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.



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.



7. TBP (yellow) binds to the DNA, using the TATA-box to position itself near the iniation site of transcription.



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.




They 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.


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.




* 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

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