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Theory of Intelligent Design, the best explanation of Origins » Origin of life » The RNA & DNA World » transfer RNA

transfer RNA

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1 transfer RNA on Fri Jun 26, 2015 10:12 am


Transfer RNA, and its biogenesis

tRNA's are very specific and complex molecules, and the " made of " follows several steps, requiring a significant number of proteins and enzymes, which are by themself also enormously complex, not only in their structure, but as well in their " made of ". So the question in the end arises : did natural processes have foresight of the end product, tRNA, to make these highly specific nano robot - like molecular machines which  remove, add and  modify  the nucleotides ? If not, how could they have arisen, since without end goal, there would be no function for them ? these enzymes are all specifically made for the production of tRNA's. And tRNA is essential for life  

Transfer RNA is an ancient molecule, central to every task a cell performs and thus essential to all life.
The enzyme is one of only two ribozymes which can be found in all kingdoms of life (Bacteria, Archaea, and Eukarya). 11

The three major RNAs involved in the flow of genetic information are messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). All these RNAs participate in the protein-synthesizing pathway in cells. tRNA has two distinct characteristics. It carries an anticodon corresponding to the mRNA codon and it binds to the corresponding amino acid in a reaction catalyzed by a specific aminoacyl-tRNA synthetase. In this sense, tRNA is a key bridging molecule between ribonucleotide information (RNA world) and peptide information (protein world). Therefore, tracing the evolution origin of tRNA molecules is likely to cast light on the processes that led to the establishment of the central dogma. 16

Of the thousands of RNAs so far identified, transfer RNA (tRNA) is the most direct intermediary between genes and proteins. Like many other RNAs (ribonucleic acids), tRNA aids in translating genes into the chains of amino acids that make up proteins. With the help of a highly targeted enzyme, each tRNA molecule recognizes and latches onto a specific amino acid, which it carries into the protein-building machinery. In order to successfully add its amino acid to the end of a growing protein, tRNA must also accurately read a coded segment of messenger RNA, which gives instructions for the exact sequence of amino acids in the protein. see here

tRNAs Are covalently modified before they exit from the Nucleus

Like most other eucaryotic RNAs, tRNAs are covalently modified before they are allowed to exit from the nucleus.Eucaryotic tRNAs are synthesized by RNA Polymerase III. Both bacterial and eucaryotic tRNAs are typically synthesized as larger precursor tRNAs, which are then trimmed to produce the mature tRNA. In addition, some tRNA precursors (from both bacteria and eucaryotes) contain introns that must be spliced out. This splicing reaction differs chemically from pre-mRNA splicing; rather than generating a lariat intermediate, tRNA splicing uses a cut-and-paste mechanism that is catalyzed by proteins See below:

Trimming and splicing both require the precursor tRNA to be correctly folded in its cloverleaf configuration. Because misfolded tRNA precursors will not be processed properly, the trimming and splicing reactions are thought to act as quality- control steps in the generation of tRNAs. All tRNAs are modified chemically—nearly 1 in 10 nucleotides in each mature tRNA molecule is an altered version of a standard G, U, C, or A ribonucleotide.Over 50 different types of tRNA modifications are known; a few are shown below:

Some of the modified nucleotides—most notably inosine, produced by the deamination of adenosine—affect the conformation and basepairing of the anticodon and thereby facilitate the recognition of the appropriate mRNA codon by the tRNA molecule. Others affect the accuracy with which the tRNA is attached to the correct amino acid.Some of the modified nucleotides—most notably inosine, produced by the deamination of adenosine—affect the conformation and basepairing of the anticodon and thereby facilitate the recognition of the appropriate mRNA codon by the tRNA molecule . Others affect the accuracy with which the tRNA is attached to the correct amino acid.[/b]

After tRNA is transcribed by RNA polymerase III as a precursor tRNA, it  must be processed into a mature tRNA

This happens through  the removal, addition and chemical modification of nucleotides. Processing for some tRNA involves 2

   1) removal of the leader sequence at the 5 prime end
   2) replacement of two nucleotides at the 3 prime end by the sequence CCA (with which all mature tRNA molecules terminate)
   3) chemical modification of certain bases and
   4) excision of an intron.

The mature tRNA is often diagrammed as a flattened cloverleaf which clearly shows the base pairing between self-complementary stretches in the molecule.

tRNA maturation in Homo sapiens

Enzymatic  complexes involved in the process: 18

CCA tRNA nucleotidyltransferase 1
Zinc phosphodiesterase ELAC protein 2

Enzymatic  complexes:
Ribonuclease P
tRNA ligase complex
tRNA-splicing endonuclease




1)The transcription product, the pre-tRNA, contains additional RNA sequences at both the 5’ and 3’-ends.   These additional sequences are removed from the transcript during processing. The additional nucleotides at the 5’-end are removed by an unusual RNA containing enzyme called ribonuclease P (RNase P)

Some tRNA precursors contain an intron located in the anticodon arm. These introns are spliced out during processing of the tRNA.

The cloverleaf structure of a single polynucleotide tRNA molecule is universally conserved among organisms. However, tRNA genes are often divided into parts on the chromosome; in bacteria, archaea, eukarya, and organelles, several tRNA genes are interrupted by various types of introns, which are removed by RNA splicing after transcription
Introns in nuclear and archaeal tRNAs are generally cleaved by tRNA-splicing endonuclease 13

tRNA splicing is a fundamental process required for cell growth and division. SEN2 is a subunit of the tRNA splicing endonuclease, which catalyzes the removal of introns, the first step in tRNA splicing 14

The tRNA splicing reaction in yeast occurs in three steps; each step is catalyzed by a distinct enzyme, which can function interchangeably on all of the substrates 15

All mature tRNAs contain the trinucleotide CCA at their 3’-end. These three bases are not coded for by the tRNA gene. Instead, these nucleotides are added during processing of the pre-tRNA transcript. The enzyme responsible for the addition of the CCA-end is tRNA nucleotidyl transferase and the reaction proceeds according to the following scheme:

tRNA +CTP --> tRNA-C + PPi (pyrophosphate)
tRNA-C +CTP --> tRNA-C-C + PPi
tRNA-C-C +ATP --> tRNA-C-C-A + PPi

Mature tRNAs can contain up to 10% bases other than the usual adenine (A), guanine (G), cytidine (C) and uracil (U). These base modifications are introduced into the tRNA at the final processing step. The biological function of most of the modified bases is uncertain and the translation process seems normal in mutants lacking the enzymes responsible for modifying the bases.

Termination signals end the transcription of RNA by RNA polymerase I and RNA polymerase III without the activity of hairpin structures as seen in prokaryotes.
mRNA is cleaved 10 to 35 base-pairs downstream of a AAUAAA sequence (which acts as a poly-A tail addition signal).

The biogenesis of mature transfer (t)RNAs in cells has a complexity that belies the elegance of their function as the adaptor molecules of protein synthesis. They are synthesized as precursors which are converted to mature tRNA molecules by a sequence of events that includes processing of their 5′ and 3′ ends, modification of a number of bases, addition of the terminal CCA residues and, in the case of intron-containing tRNAs, splicing 1

Transfer RNAs (tRNAs) play an important role linking mRNA and amino acids during protein biogenesis. Four types of tRNA genes have been identified in living organisms. However, the evolutionary origin of tRNAs remains largely unknown.   Given their central role in life and their high level of conservation, tRNAs have stimulated extensive interest in their origin and evolution

this enzyme alone is like a small cogwheel in a watch in a a complex clockwork mechanism. If you take it away, the whole mechanism of protein synthesis ceases to exist. No RNase, no proteinsynthesis, no life....... As with RNase, the Protein production needs hundreds, well, probably thousands of intricate fine tuned parts, all doing their function in a precise way, and if you take away just one small apparently irrelevant part, bye bye proteins, bye bye life. These enzyme subparts have no use by their own, but only if correctly inserted in the intricate holoenzyme complexes exercising precisely their function.........

further readings :

Molecular biology of the transfer RNA revisited

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2 Transcription of tRNA on Thu Jul 02, 2015 11:36 am


Transcription of tRNA

tRNAs are transcribed by RNA polymerase III as pre-tRNAs in the nucleus. RNA polymerase III
recognizes two internal promoter sequences (A-box B internal promoter) inside tRNA genes.
The first promoter begins at nucleotide 8 of mature tRNAs and the second promoter is located 30-60
nucleotides downstream of the first promoter. The transcription terminates after a stretch of
four or more thymidines.12

RNA polymerase III is responsible for the production of pre-tRNAs, 5SrRNA and other small RNAs. 2

tRNA Synthesis & Processing 4

RNA polymerase III is responsible for the production of pre-tRNAs, 5SrRNA and other small RNAs. 2

RNA polymerase III:  The promoters for RNA polymerase III vary in structure but the ones for tRNA genes and 5S rRNA genes are located entirely downstream of the startpoint, within the transcribed sequence.  In tRNA genes, about 30-60 base-pairs of DNA separate promoter elements; in 5S rRNA genes, about 10-30 base-pairs promoter elements.

General transcription factors and the polymerase undergo a pattern of  sequential binding to initate transcription of nuclear genes.
1) TFIID binds to the TATA box followed by
2) the binding of TFIIA and TFIIB.
3) The resulting complex is now bound by the polymerase, to which TFIIF has already attached.
4) The initiation complex is completed by the addition of TFIIE, TFIIJ, and TFIIH.
5) After an activation step requiring ATP-dependent phosphorylation of the RNA polymerase molecule, the polymerase can initiate transcription at the startpoint.

The TATA-binding protein (TBP) is a subunit of the TFIID and plays a role in the activity of the  RNA polymerase III transcription.
TBP is also essential for transcription of TATA-less genes.
TBP differs from most DNA-binding proteins in that it interacts with the minor groove of DNA, rather than the major groove and imparts a sharp bend to the DNA.
The TBP is highly conserved.
When TBP is bound to DNA, other transcription-factor proteins can interact with the convex surface of the TBP saddle.
TBP is required for transcription initiation on all types of eukaryotic promoters.

In order to make tRNA, 1. the genetic information stored in DNA is required, as well as the information to make the machines that process the newly synthesized and unfinished pre-tRNAs strand, so as the RNA polymerase III complex , together with all subunits, co-factors, transcription factors etc. to transcribe DNA into pre-tRNAs, beside ATP, the fuel to make all happen. Well over one hundred sub units are required in such a complex process, that science even today does not fully understand the details.  That is a irreducible complex, and interdependent sophisticated and highly complex system,  which requires a enormous amount of complex, specified, coded information stored in DNA.


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3 CCA tRNA nucleotidyltransferase 1 on Sat Jul 04, 2015 4:40 pm


CCA tRNA nucleotidyltransferase 1


Isoform 1: Adds and repairs the conserved 3'-CCA sequence necessary for the attachment of amino acids to the 3' terminus of tRNA molecules, using CTP and ATP as substrates. Ref.9
Isoform 2: Adds 2 C residues (CC-) to the 3' terminus of tRNA molecules instead of a complete CCA end as isoform 1 does (in vitro).

Recognition of 3' end without CCA by tRNA nucleotidyltransferase in Homo sapiens

Addition of CCA sequence to 3'end of intron-containing pre-tRNA in Homo sapiens

Transfer RNA nucleotidyltransferases (CCA-adding enzymes) are responsible for the maturation or repair of the functional 3′ end of tRNAs by means of the addition of the essential nucleotides CCA. However, it is unclear how tRNA nucleotidyltransferases polymerize CCA onto the 3′ terminus of immature tRNAs without using a nucleic acid template. 1

The acylation of all tRNAs with an amino acid occurs at the terminal ribose of a 3' CCA sequence.
The CCA sequence is added to the tRNA precursor by stepwise nucleotide addition performed by a single enzyme that is ubiquitous in all living organisms.
Although the enzyme has the option of releasing the product after each addition, it prefers to stay bound to the product and proceed with the next addition.  4

tRNA-nucleotidyltransferases: Highly unusual RNA polymerases with vital functions 3

tRNA-nucleotidyltransferases are fascinating and unusual RNA polymerases responsible for the synthesis of the nucleotide triplet CCA at the 3′-terminus of tRNAs. As this CCA end represents an essential functional element for aminoacylation and translation, these polymerases (CCA-adding enzymes) are of vital importance in all organisms.

CCA-adding enzymes obviously can count until three: after the addition of three nucleotides, the polymerization reaction is efficiently stopped Additionally, and most interestingly, the CCA-adding enzymes recognize if nucleotides are previously added to a tRNA primer and incorporate then only the missing ones, completing thereby the CCA triplet. A tRNA that carries already the first C residue of the CCA terminus is elongated only by one C and one A, while on a tRNA ending with CC, only the terminal A residue is added. This feature shows that CCA-adding enzymes are not only responsible for the de novo synthesis of CCA ends but have an important maintenance and repair function for tRNA ends. This stringent sequence and length control of the tRNA CCA end reflects the recognition requirements for aminoacylation and translation. ( this is amazing. How did it " learn "  that feat ? trial and error ?  )

Furthermore, positioning in the ribosome during translation and even peptide release from the ribosome depend on an intact CCA end, which is critical for water coordination and efficient hydrolysis of the ester bound translation product

These facts indicate that an accurate CCA end participates, beyond simple recognition and binding, as an integral part in several reaction mechanisms and is therefore of vital importance for the cell.

Surprisingly, these polymerases with such unusual features evolved twice in evolution, leading to classes 1 and 2 CCA-adding enzymes

Convergence is evidence against evolution, and the author supposes evolution prior the existence of a replicating cell 5

While class 1 is exclusively found in archaea, class 2 tRNA-nucleotidyltransferases are present in eukaryotes and bacteria, where they fulfill identical functions.

Structural organization of classes 1 and 2 CCA-adding enzymes. While both enzyme versions have a hook-like shape of similar size, the allocation of secondary structure elements in neck, body and tail domains are quite different. In class 1 enzymes, these regions contain alpha-helical as well as beta-sheet elements. Class 2, on the other hand, has exclusively alpha-helical structures in these domains. The catalytic cores, located in head and neck domains of both enzyme versions, are indicated by the grey arrows. The rainbow color bar represents the consecutive protein regions from N- (blue) to C-terminus (red).

One of the most fascinating aspects of both classes of tRNA-nucleotidyltransferases is the fact that CCA-addition does not require an external nucleic acid as a template – somehow these enzymes “know” when to incorporate which nucleotide.

Indeed. Isn't that a magnificient example and evidence of design ?

Crystal structures of both classes 1 and 2 enzymes revealed a set of highly conserved amino acid residues located in the single nucleotide binding pocket that interact with the incoming nucleotide by forming Watson/Crick-like hydrogen bonds

So these enzymes do not only " know " when to incorporate which nucleotide, but also " know " how to bind each nucleotide to the next through hydrogen bonds..... amazing.

The 3′-terminal CCA nucleotide sequence  of transfer RNA is essential for amino acid attachment and interaction with the ribosome during protein synthesis. The CCA sequence is synthesized de novo and/or repaired by a template-independent RNA polymerase, ‘CCA-adding enzyme’, using CTP and ATP as substrates

tRNA nucleotidyltransferase

In eukaryotes, multiple forms of tRNA nucleotidyltransferases are synthesized from a single gene and are distributed to different subcellular compartments in the cell. There are multiple in-frame start codons which allow for the production of variant forms of the enzyme containing different targeting information predominantly found in the N-terminal sequence of the protein 2


CCA addition :

Elucidation of the role of the CCA enzyme in the cellular network of tRNA quality control and the identities of the RNases accompanying the CCA enzyme constitute new questions that warrant active investigation.

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4 Zinc phosphodiesterase on Sun Jul 05, 2015 8:08 am


Zinc phosphodiesterase ELAC protein 2

Function 1

Zinc phosphodiesterase, which displays some tRNA 3'-processing endonuclease activity. Probably involved in tRNA maturation, by removing a 3'-trailer from precursor tRNA.

Catalytic activity

Endonucleolytic cleavage of RNA, removing extra 3' nucleotides from tRNA precursor, generating 3' termini of tRNAs. A 3'-hydroxy group is left at the tRNA terminus and a 5'-phosphoryl group is left at the trailer molecule.

Processing of 3' end by RNase Z in Homo sapiens

Processing of 3' end by RNase Z in Homo sapiens


Recognition of 3' end by RNase Z in Homo sapiens


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5 Ribonuclease P on Sun Jul 05, 2015 8:13 am


Ribonuclease P

Structure of Ribonuclease P (Homo sapiens):

Human nuclear RNase P consists of 10 Protein subunits and one RNA subunit.

Protein components:

Ribonuclease P protein subunit p14
Ribonuclease P protein subunit p20
Ribonuclease P protein subunit p21
Ribonuclease P protein subunit p25
Ribonuclease P protein subunit p29
Ribonuclease P protein subunit p30
Ribonuclease P protein subunit p38
Ribonuclease P protein subunit p40
Ribonucleases P/MRP protein subunit POP1
Ribonuclease P/MRP protein subunit POP5

RNA components:


Reactions in which Ribonuclease P is involved:

Recognition of 5' end by RNase P in Homo sapiens

Processing of 5' end by RNase P in Homo sapiens

Ribonuclease P (EC, RNase P) is a type of ribonuclease which cleaves RNA. RNase P is unique from other RNases in that it is a ribozyme – a ribonucleic acid that acts as a catalyst in the same way that a protein based enzyme would. Its function is to cleave off an extra, or precursor, sequence of RNA on tRNA molecules. 6

RNase P is one of two known multiple turnover ribozymes in nature (the other being the ribosome), the discovery of which earned Sidney Altman and Thomas Cech the Nobel Prize in Chemistry in 1989

Bacterial RNase P has two components: an RNA chain, called M1 RNA, and a polypeptide chain, or protein, called C5 protein

Sidney Altman of Department of Molecular, Cellular and Developmental Biology Yale University, writes in his paper: Ribonuclease P following:

It seems unreasonable that ribosomes, with three RNAs and about 50 proteins, would exist without less complex RNPs having come into existence first and having persisted
throughout evolution. 5

And for what reason would Ribonuclease P have evolved to make tRNA, if tRNA would provide no function, unless embedded in the Ribosome used as physical link between the nucleotide sequence of nucleic acids (DNA and RNA) and the amino acid sequence of proteins ? Furthermore, its second component , C5 protein, is made not made of RNA, but amino acids, that is, tRNA are required to make the machine that makes tRNA. Another example of catch22 situation  

In eukaryotes, such as humans and yeast, most RNase P consists of an RNA chain that is structurally similar to that found in bacteria  as well as nine to ten associated proteins (as opposed to the single bacterial RNase P protein, C5)

In archaea and eukarya the enzyme has evolved an increasingly more complex protein composition, whilst retaining a structurally related RNA subunit. The reasons for this additional complexity are not currently understood. Furthermore, the eukaryotic RNase P has evolved into several different enzymes including a nuclear activity, organellar activities, and the evolution of a distinct but closely related enzyme, RNase MRP, which has different substrate specificities, primarily involved in ribosomal RNA biogenesis. 11

In eukaryotes, RNase P appears to have split into a number of distinct activities, including diverse pre-tRNA cleavage activities found in eukaryotic organelles, such as mitochondria and chloroplasts

Eukaryotes possess both a nuclear RNase P, and another related essential endoribonuclease, RNase MRP, which is found only in eukaryotes and cleaves at least three distinct substrates.

The majority of RNase MRP is located in the nucleus where it processes precursor ribosomal RNA (pre-rRNA)

Other characterized functions are the processing of mitochondrial RNAs (mtRNAs) leading to the generation of primers for mitochondrial DNA (mtDNA) replication

RNase MRP also plays a role in the degradation of the mRNA of a B-type cyclin, an activity which appears to be important for cell cycle progression

A second issue of interest to biochemists and “evolutionists” is the puzzle presented by the different compositions of RNase P from eubacteria (one catalytic RNA and one protein subunit) and from eukaryotes (one RNA subunit and several protein subunits: no understanding yet of which subunit is responsible for catalysis).The complexity of eukaryotic RNase P presents the questions of why there are so many subunits and what relation this complexity might have to the intracellular localization of the enzyme, possible isoforms , and its relation to other enzymes with which it might interact in vivo. RNase P in organelles, e.g., chloroplasts and mitochondria, present their own complexities in terms of the origin and function of their subunits . More generally, the role of RNase P in the regulation of tRNA and rRNA biosynthesis in both prokaryotes and eukaryotes has not yet been fully explored.

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that cleaves precursor tRNA transcripts to give mature 5′ ends. RNase P in eubacteria has a large, catalytic RNA subunit and a small protein subunit that are required for precursor tRNA cleavage in vivo. Although the eukaryotic holoenzymes have similar, large RNA subunits, previous work in a number of systems has suggested that the eukaryotic enzymes require a greater protein content. We have purified the Saccharomyces cerevisiae nuclear RNase P to apparent homogeneity, allowing the first comprehensive analysis of an unexpectedly complex subunit composition.

Function of Rnase P

In E. coli, RNase P processes not only ptRNAs, but also, at the very least, the precursors to 4.5S RNA

Signal recognition particle RNA 4.5S RNA   7

The signal recognition particle RNA, also known as  4.5S RNA, is the RNA component of the signal recognition particle (SRP) ribonucleoprotein complex.SRP is a universally conserved ribonucleoprotein that directs the traffic of proteins within the cell and allows them to be secreted. The signal recognition particle, SRP RNA, together with one or more SRP proteins contributes to the binding and release of the signal peptide. 8

A signal peptide (sometimes referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) is a short (5-30 amino acids long) peptide present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway

In prokaryotes, signal peptides direct the newly synthesized protein to the SecYEG protein-conducting channel, which is present in the plasma membrane.

Protein Localization in the Cell 9

Proteins are the workhorses of the typical cell. Originally coded for by DNA and composed of a sequence of amino acids, proteins serve a variety of roles including binding other molecules and/or transporting them, serving as 'switches' for cellular activity, catalyzing chemical reactions, and even acting as structural building blocks. It should come as no surprise then that in order to accomplish all these goals, proteins are needed in a variety of locations both inside and outside of the cell. Therefore, a variety of mechanisms have evolved in order to correctly localize proteins, helping to guide them from their point of synthesis (at the ribosome) to their destination.One such set of mechanisms is the secretory pathway. Also ubiquitous in biological systems are membranes, barriers between the cell and the outside world. Membranes also serve to divide compartments within some cells (those of eukaryotes). In order for proteins to get to where they are needed inside these compartments or outside the cell, they need a way to cross the membrane without rupturing it at the same time. The secretory pathway consists in part of a channel for just this purpose: the translocon

So, interestingly, PNase processes various  completely different molecules, namely tRNA, and the precursors of the signal recognition particle, beside others. How could that be explained from a naturalistic/evolutionary standpoint ? Lets assume, evolution were a driving force at this stage ( which it couldn't be ), for what reason would such a enzyme involve in the manufacturing process of two completely different molecular parts ? From a naturalistic standpoint, thats hard to grasp. From a intelligent design standpoint however, that makes perfectly sense. Designers sometimes create machines, involved in the manufacturing of different parts for various reasons. BTW. Pnase P would have no function, until fully developed, and working in concert and synchronisation with other manufacturing machines inside the cell, to produce molecular parts , required for the functioning of the cell.

Ribonuclease P(EC, RNase P) is a type of ribonuclease which cleaves RNA. RNase P is unique from other RNases in that it is a ribozyme – a ribonucleic acid that acts as a catalyst in the same way that a protein based enzyme would. Its function is to cleave off an extra, or precursor, sequence of RNA on tRNA molecules.


further readings :

Ribonuclease P (RNase P) has been hitherto well known as a catalytic ribonucleoprotein that processes the 5' leader sequence of precursor tRNA. Recent studies, however, reveal a new role for nuclear forms of RNase P in the transcription of tRNA genes by RNA polymerase (pol) III, thus linking transcription with processing in the regulation of tRNA gene expression. However, RNase P is also essential for the transcription of other small noncoding RNA genes, whose precursor transcripts are not recognized as substrates for this holoenzyme. Accordingly, RNase P can act solely as a transcription factor for pol III, a role that seems to be conserved in eukarya.

In 1989, Sidney Altman and Thomas R. Cech shared the Nobel Prize in Chemistry for their discovery of catalytic properties of RNA. Cech was studying the splicing of RNA in a unicellular organism called Tetrahymena thermophila. He found that the precursor RNA could splice in vitro in the absence of proteins. Altman studied ribonuclease P (RNase P), a ribonucleoprotein that is a key enzyme in the biosynthesis of tRNA. RNase P is an RNA processing endonuclease that specifically cleaves precursors of tRNA, releasing 5' precursor sequences and mature tRNAs. RNase P is involved in processing all species of tRNA and is present in all cells and organelles that carry out tRNA synthesis. What follows is a personal recollection by Altman of how he came to study this remarkable enzyme.

Ribonuclease P (RNase P) is an endonuclease involved in processing tRNA. It contains both RNA and protein subunits and occurs in all three domains of life: namely, Archaea, Bacteria and Eukarya. The RNase P RNA subunits from bacteria and some archaea are catalytically active in vitro, whereas those from eukaryotes and most archaea require protein subunits for activity. RNase P has been characterized biochemically and genetically in several systems, and detailed structural information is emerging for both RNA and protein subunits from phylogenetically diverse organisms. In vitro reconstitution of activity is providing insight into the role of proteins in the RNase P holoenzyme. Together, these findings are beginning to impart an understanding of the coevolution of the RNA and protein worlds.

Catalytic complexes of nuclear ribonuclease P (RNase P) ribonucleoproteins are composed of several protein subunits that appear to have specific roles in enzyme function in tRNA processing. This review describes recent progress made in the characterization of human RNase P, its relationship with the ribosomal RNA processing ribonucleoprotein RNase MRP, and the unexpected evolutionary conservation of its subunits. A new model for the biosynthesis of human RNase P is presented, in which this process is dynamic, transcription-dependent, and implicates functionally distinct nuclear compartments in tRNA biogenesis.

more :

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6 tRNA ligase complex on Sun Jul 05, 2015 8:29 am


tRNA ligase complex

Protein components: 1

tRNA-splicing ligase RtcB homolog
ATP-dependent RNA helicase DDX1
Protein FAM98B
UPF0568 protein C14orf166
Protein archease

Reactions in which tRNA ligase complex is involved:

Recognition of spliced pre-tRNA by tRNA ligase complex in Homo sapiens

Ligation of exon ends in Homo sapiens

Diversity and roles of (t)RNA ligases.

The discovery of discontiguous tRNA genes triggered studies dissecting the process of tRNA splicing. As a result, we have gained detailed mechanistic knowledge on enzymatic removal of tRNA introns catalyzed by endonuclease and ligase proteins. In addition to the elucidation of tRNA processing, these studies facilitated the discovery of additional functions of RNA ligases such as RNA repair and non-conventional mRNA splicing events. Recently, the identification of a new type of RNA ligases in bacteria, archaea, and humans closed a long-standing gap in the field of tRNA processing. This review summarizes past and recent findings in the field of tRNA splicing with a focus on RNA ligation as it preferentially occurs in archaea and humans. In addition to providing an integrated view of the types and phyletic distribution of RNA ligase proteins known to date, this survey also aims at highlighting known and potential accessory biological functions of RNA ligases.

The human tRNA ligase complex 3′–5′ RNA ligation appears to be the prevalent human tRNA splicing pathway  and relies on HSPC117 as the essential ligase component Human HSPC117, together with the proteins DDX1, CGI-99, FAM98B, and ASW, forms a stable complex of about 200 kDa

HSPC117 is the essential subunit of a human tRNA splicing ligase complex.

Splicing of mammalian precursor transfer RNA (tRNA) molecules involves two enzymatic steps. First, intron removal by the tRNA splicing endonuclease generates separate 5' and 3' exons. In animals, the second step predominantly entails direct exon ligation by an elusive RNA ligase. Using activity-guided purification of tRNA ligase from HeLa cell extracts, we identified HSPC117, a member of the UPF0027 (RtcB) family, as the essential subunit of a tRNA ligase complex. RNA interference-mediated depletion of HSPC117 inhibited maturation of intron-containing pre-tRNA both in vitro and in living cells. The high sequence conservation of HSPC117/RtcB proteins is suggestive of RNA ligase roles of this protein family in various organisms.

Transfer RNAs (tRNAs) are essential adaptor molecules in the translation of the genetic transcript into proteins. During their posttranscriptional maturation (1), intron-containing tRNA precursor transcripts (pre-tRNAs) undergo splicing, which is accomplished by a specialized endonuclease that excises the intron (2, 3) and a ligase that joins the resulting exon halves

Transfer RNAs (tRNA) are transcribed as precursor transcripts and are subjected to a series of posttranscriptional processing events before they are matured to fulfill their biological functions Sequence analysis of tRNA genes in mammals, revealed the existence of tRNA genes disrupted by intervening sequences. Intron harboring tRNA genes are now known to occur in the genomes of organisms from all three domains of life. After the discovery of intron-containing tRNAs, the mechanistic features of tRNA splicing were extensively studied. Eukaryal pre-tRNA transcripts undergo enzymatic splicing. The latter achieves intron removal by endoribonucleolytic cleavage and subsequent ligation rather than by two consecutive transesterification events as employed by self-splicing introns or the spliceosome.


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7 tRNA-splicing endonuclease on Sun Jul 05, 2015 8:38 am


tRNA-splicing endonuclease

Protein components:

tRNA-splicing endonuclease subunit Sen54
tRNA-splicing endonuclease subunit Sen2
tRNA-splicing endonuclease subunit Sen15
tRNA-splicing endonuclease subunit Sen34

Reactions in which tRNA-splicing endonuclease is involved:

Recognition of intron-containing tRNA by tRNA-splicing endonuclease in Homo sapiens

Identification of a human endonuclease complex reveals a link between tRNA splicing and pre-mRNA 3' end formation.

Both human endonuclease complexes are associated with pre-mRNA 3' end processing factors. Furthermore, siRNA-mediated depletion of SEN2 exhibited defects in maturation of both pre-tRNA and pre-mRNA. These findings demonstrate a link between pre-tRNA splicing and pre-mRNA 3' end formation, suggesting that the endonuclease subunits function in multiple RNA-processing events.

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8 The enigma of ribonuclease P evolution on Sun Jul 05, 2015 5:49 pm


The enigma of ribonuclease P evolution

Split tRNA genes are encoded at different loci of C. maquilingensis genome. The tRNA fragments (A-E) are individually transcribed and assembled via trans-splicing based on the leader sequences (black) located at one or both ends. Sequences A and D are used multiple times in a different combination, as in a jigsaw puzzle.

In addition, a number of split tRNA genes have been recently found in the Desulfurococcales branch of archaea, expanding the population of split genes to diverse archaeal species. Examination of their gene arrangement combined with phylogenetic analysis has indicated that split tRNAs was a late acquisition, most likely created through local genome rearrangement. This means that split tRNAs in the archaeal genome might not be direct homologs but rather analogs of ancestral tRNAs

The simplest interpretation is that RNase P has an ‘RNA-alone’ origin and progenitors of Bacteria and Archaea diverged very early in evolution and then pursued completely different strategies in the recruitment of protein subunits during the transition from the ‘RNA-alone’ to the ‘RNA-protein’ state of the enzyme.’ 2

The author writes about recruitment and strategies. Its interesting that he atributes mental and conscient activities to chemical processes and reactions. But as such, they have no end goal, so how does it make sense to write in these terms ? Furthermore, recruitment of what ? of extant subunits ? were they readily available to choose from in the surrounding ?  how could RNase know which ones to select  and  how to incorporate them correctly in its system ? Is that not one more nice example of pseudo science ?

As Luskin writes : When certain biologists discuss the early stages of life there is a tendency to think too vaguely. They see a biological wonder before them and they tell a story about how it might have come to be. They may even draw a picture to explain what they mean. Indeed, the story seems plausible enough, until you zoom in to look at the details. I don't mean to demean the intelligence of these biologists. It's just that it appears they haven't considered things as completely as they should. Like a cartoon drawing, the basic idea is portrayed, but there is nothing but blank space where the profound detail of biological processes should be.

Would these five conditions not have to be met in order to recruit and insert the subunits into the system ?

C1: Availability. Among the parts available for recruitment to form the system, there would need to be ones capable of performing the highly specialized tasks of individual parts, even though all of these items serve some other function or no function.

C2: Synchronization. The availability of these parts would have to be synchronized so that at some point, either individually or in combination, they are all available at the same time.

C3: Localization. The selected parts must all be made available at the same ‘construction site,’ perhaps not simultaneously but certainly at the time they are needed.

C4: Coordination. The parts must be coordinated in just the right way: even if all of the parts of a system are available at the right time, it is clear that the majority of ways of assembling them will be non-functional or irrelevant.

C5: Interface compatibility. The parts must be mutually compatible, that is, ‘well-matched’ and capable of properly ‘interacting’: even if sub systems or parts are put together in the right order, they also need to interface correctly.

( Agents Under Fire: Materialism and the Rationality of Science, pgs. 104-105 (Rowman & Littlefield, 2004). HT: ENV.)

This is in contrast to the recent proposal that the archaeal/eukaryal-type RNase P proteins might represent the ancestral type of RNase P proteins, and the bacterial C5-type proteins are the result of a more recent innovative evolutionary change. However, we note that such a scenario would have required a complex sequence of evolutionary changes. It implies that the progenitor of extant bacteria gave up its RNase P holoenzyme architecture involving the typically four archaeal-type protein subunits  and simultaneously invented the single C5-type protein subunit. The divergent outcome of the recruitment of protein subunits during the transition period from the ‘RNA-alone

Its interesting that the author does not note that the same complexity would imply with  a  bottom - up  evolutionary transition, that is from simpler bacterial, to eukaryotic holoenzymes. The ' invention ' of C5 type protein subunit had to happen anyway. And there is nowhere in the paper a credible detailled explanation of how that could possibly have happened. The problems with these naturalistic explanations are manyfold. First of all, the authors mention evolution as if that could be a driving force at this stage. It isnt. Secondly, and that is a severe problem : for what reason would such a enzyme evolve ? it would have by its own no function. It adquires only its function when actually ready to do its job, and the unfinished tRNA strand is ready to be cleaved. Its interesting that the authors of evolutionary papers never raise these evident concerns. Ever asked, why ?

MOLECULAR FOSSIL Crystal structure shows how RNA, one of biology’s oldest catalysts, is made1

“RNA is an ancient molecule, but it is pretty sophisticated,” said Alfonso Mondragón, professor of molecular biosciences in the Weinberg College of Arts and Sciences. He led the research. “Our crystal structure shows that it has many of the properties we ascribe to modern molecules. RNA is a catalyst that has much of the versatility and complexity of modern-day proteins.”

For billions of years and still to this day, the function of RNase P -- found in nearly all organisms, from bacteria to humans -- has been to cleave transfer tRNA. If the tRNA is not cleaved, it is not useful to the cell.

“We knew this important chemistry happened, that RNA acts as a catalyst, but we didn’t know exactly how until now,” Mondragón said. “We now have a better understanding of how RNA works.”

RNase P is formed by a large RNA core plus a small protein, illustrating the evolutionary shift from an RNA world toward a protein-dominated world.

Well, no. It illustrates that a supposed transition from RNA to DNA could not occure. How was the protein made ? If there was a RNA world, but no DNA, then proteins could not be produced yet. Quite obvious. But not to the author, which remains suspisciously  silent about this fact. Furthermore , if PNase had only two subunits, it would already be a candidate to be considered irreducible complex, since both subunits are required to cleave tRNA.

The protein helps recognize the tRNA, but most of the recognition occurs through RNA-RNA interactions involving shape complementarity and also base pairing.

The structure shows that once RNase P recognizes tRNA, it docks and, assisted by metal ions, cuts one chemical bond.

This is a stepwise, orderly, precise process, where the enzyme " knows " exactly where to cleave with a precise target. How could such a function have arisen ? trial and error ? why at all trial and error ? Or had chemicals a end goal ? or the goal of " survival of the fittest " , despite the fact that they are not alive ? if the enzyme cleaved too much or too less, no function of the tRNA..... so its function had to be programmed correctly in the genome right from the start, otherwise, well, no life.... my contemption is once more that naturalistic explanations are completely inadequate to explain this sophisticated mechanism in question. While a designer, which had the intention to make life, could have well invented the process, and set it up. So ID is in my view a far better and more adequate explanation than random chance or chemical reactions.

This matures the tRNA, producing a smaller RNA molecule that now can contribute to fundamental processes in the cell. The RNA-based enzyme does this over and over, cutting each tRNA in exactly the same place every time.

“The discovery nearly 30 years ago that RNA molecules can have a catalytic function raised the idea that maybe RNA was the first molecule,” Mondragón said. “Our work reinforces this notion of the existence of an RNA world when life first began.”

The catalytic function here described is extremely sophysticated, precise, and programmed. By no means it permits a leap of faith to infer the transition from a RNA to a DNA world.

Last edited by Admin on Mon Jul 06, 2015 9:53 am; edited 7 times in total

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9 Re: transfer RNA on Sun Jul 05, 2015 5:50 pm


tRNA Biology in Mitochondria

Mitochondria are the powerhouses of eukaryotic cells. They are considered as semi-autonomous because they have retained genomes inherited from their prokaryotic ancestor and host fully functional gene expression machineries. These organelles have attracted considerable attention because they combine bacterial-like traits with novel features that evolved in the host cell. Among them, mitochondria use many specific pathways to obtain complete and functional sets of tRNAs as required for translation. In some instances, tRNA genes have been partially or entirely transferred to the nucleus and mitochondria require precise import systems to attain their pool of tRNAs. Still, tRNA genes have also often been maintained in mitochondria. Their genetic arrangement is more diverse than previously envisaged. The expression and maturation of mitochondrial tRNAs often use specific enzymes that evolved during eukaryote history. For instance many mitochondria use a eukaryote-specific RNase P enzyme devoid of RNA. The structure itself of mitochondrial encoded tRNAs is also very diverse, as e.g., in Metazoan, where tRNAs often show non canonical or truncated structures. As a result, the translational machinery in mitochondria evolved adapted strategies to accommodate the peculiarities of these tRNAs, in particular simplified identity rules for their aminoacylation. Here, we review the specific features of tRNA biology in mitochondria from model species representing the major eukaryotic groups, with an emphasis on recent research on tRNA import, maturation and aminoacylation.


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Evolution of transfer RNA and the origin of the translation system

The origin of the translation system is at the center of discussions about the evolution of biological systems.

In this context, molecules of transfer RNA (tRNA) are highlighted due to its ability to convey the information contained in nucleic acids with the functional information contained in the proteins. Despite many characteristics shared among tRNAs in various organisms, suggesting a monophyletic origin for this group of molecules, recent discussions have proposed a polyphyletic origin for this group, thus indicating that the shared features are products of evolutionary convergence

A polyphyletic  group is characterized by one or more homoplasies: phenotypes which have converged or reverted so as to appear to be the same but which have not been inherited from common ancestors. Alternatively, polyphyletic is used to describe multiple ancestral sources regardless of convergence.

Discussions on the initial emergence of the acceptor arm or anticodon arm remain open.

Sun and Caetano-Anollés (2008) proposed from structural analysis that the acceptor arm could have arisen first and the anticodon loop is a later event in the evolutionary history of tRNAs.

If that were the case, tRNA would have lost its function.

According to the data discussed, these molecules have had this function since the beginning of the formation and organization of biological systems.

The similarity observed between the tRNA molecules and ribosomal RNAs, especially the PTC, suggest the importance of the tRNAs molecules in the assembly of the translation system, which certainly was a key event for the emergence of life on Earth.

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