# First RNA polymerase-mRNA

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We know that RNA polymerase produces mRNA by reading DNA strand. Which enzyme produces the first RNA polymerase if other RNA polymerases are synthesized in the same way like other enzymes(proteins) ?

The RNA world hypothesis states that self-replicating RNA (that is, an autocatalytic RNA polymerase) was the first form or precursor of life. So, in that context, your question is basically asking how life originated. The obvious answer is that we don't know (currently anyways), but I'm going to take this opportunity to describe a few really neat experiments which might give you some insight over a possibly contributing mechanism of how self-replicating molecules may have developed. I won't go over the Miller-Urey or Oró experiments, but let's assume that the early Earth contained organic molecules (especially nucleotides for our purposes). It's also important to keep in mind the immense time scales over which these processes occurred (the Earth is ~4.5 billion years old).

Anyways, let's talk about in vitro evolution, also known as systematic evolution of ligands by exponential enrichment (SELEX). SELEX is a procedure whereby an initial library of random RNA sequences (flanked by known sequences to be used as primer sites) undergoes a selective process followed by amplification and mutation of the selected molecules. This is repeated several times. As an example, this process can be used to create aptamers, which are small RNA molecules which bind a specific ligand. A pool of random RNA is passed over a column containing the immobilized ligand. By chance, some RNA will have some amount of affinity to the ligand. Poorly bound RNA is washed off non-stringently and then slightly better bound RNA is eluted. This RNA is reverse transcribed and amplified by PCR. The PCR primers contain the T7 RNAP promoter which allows transcription of the cDNA to produce a new RNA pool. Somewhere in this regeneration process, mutations are introduced so that the new pool contains randomly modified copies of the RNA which had some affinity. Through repeated steps of increasing stringency, an aptamer with high affinity can be developed.

(Ellington, 1994); Outline of the SELEX process.

So how does this apply to the origin of the first RNA polymerase? It just so happens that Bartel and Szostak (1993) used in vitro selection to produce a ribozyme (a catalytic RNA molecule). This ribozyme was a ligase, that is it catalyzed the formation of a phosphodiester bond between two RNA molecules that are aligned on a template.

(Bartel and Szostak, 1993); Methodology of the selection used to generate a ribozyme ligase.

While the ribozyme created was not autocatalytic, the experiment had implications on the origin of self-replicating RNA and life in general, which were discussed in the paper:

The ribozymes that we have isolated from a pool of random sequences catalyze a chemical transformation similar to that catalyzed by polymerases. Their abundance in the random-sequence pool is therefore relevant to the hypothesis that life began with an RNA replicase that originated from prebiotically synthesized random-sequence RNA. We detected about 65 sequences (Fig. 8, pools 3 and 4) capable of carrying out a particular ligation reaction in a pool of more than $$10^{15}$$ initial sequences, or a frequency of occurrence of one in about $$2*10^{13}$$ sequences.

[… ]

Presumably, sequences capable of acting as efficient template-directed RNA polymerases are more rare than this, and sequences capable of acting as an RNA replicase are even more rare. Evidently, catalysts of moderate activity could arise spontaneously from relatively small quantities of RNA (and perhaps related polynucleotides); however, a catalyst with the activity, accuracy, and dual functionality (enzyme and template) of an RNA replicase would be so rare that it could only arise spontaneously and in a single step from a truly enormous amount of RNA. The problem is compounded because two such sequences would be required for autocatalytic replication to begin, one to act as the polymerase and a second to act as the template. Therefore, an RNA replicase could only have arisen from primordial sequence pools that were not truly random. Joyce and Orgel anticipate this difficulty and propose that nonenzymatic, template-copying reactions may have had a dual role in generating primordial sequence pools that are more likely to give rise to a replicase (21). The initial pool may have been biased in favor of local secondary structure by a mechanism involving intermittent use of intramolecular sequence as the template. In addition, some sort of nonenzymatic catalysis was probably necessary for the initial copying of the replicase sequence to generate a molecule that could be used as a template, the copying of which by the replicase would generate more replicase molecules, thus initiating the autocatalytic explosion of life.

[… ]

The ribozymes that we have selected provide a new starting point for the evolution or design of RNAs with RNA polymerase and replicase activity. A series of issues including sequence-independent primer-template binding, the use of mononucleotide triphosphates as substrates, fidelity, and product-template dissociation now need to be addressed before an RNA replicase activity, an activity that has probably been extinct for over 3 billion years, can be fully resuscitated.

Johnston et al. (2001) took this experiment even further: starting with a pool of slightly mutated ribozyme ligases developed in a previous SELEX experiment, they were able to generate a ribozyme polymerase. This polymerase was able to catalyze the addition of nucleotides to a growing oligonucleotide chain up to 14 bases long and based off of an RNA template. The polymerase was also quite accurate.

(Johnston et al., 2001); Excerpts from the in vitro evolution of a ribozyme polymerase.

However, the authors are skeptical of this being the sole mechanism behind the rise of an autocatalytic ribozyme:

How could general polymerase activity have arisen on early Earth? If emergence of the first RNA replicase ribozyme coincided with the origin of life, it would have had to arise in a single step from prebiotically synthesized RNA, without the benefit of Darwinian evolution. Our shortest construct retaining activity was 165 nt, with about 90 nt involved in important Watson-Crick pairing and at least another 30 critical nucleotides (23). Ribozymes with the efficiency, accuracy, and other attributes of an RNA replicase might have to be even larger than this. However, current understanding of prebiotic chemistry argues against the emergence of meaningful amounts of RNA molecules even a tenth this length (1). This difficulty is anticipated by those who propose that life, and Darwinian evolution, began before RNA. Some speculate that in this "pre-RNA world," life was based on an RNA-like polymer, yet to be identified, that possessed the catalytic and templating features of RNA but also a more plausible prebiotic synthesis (1). The pre-RNA life forms presumably later developed the ability to synthesize RNA, facilitating the emergence of an RNA replicase ribozyme, which in turn enabled the transition to the RNA world.

There are other, similar experiments where people have made self-replicating ligases (Paul and Joyce, 2002) or a system of two, cross replicating ribozymes (Lincoln and Joyce, 2009). While the question still remains unanswered, perhaps from all of this you can get a sense of the plausibility.

References:

Bartel DP, Szostak JW. 1993. Isolation of New Ribozymes from a Large Pool of Random Sequences. Science. 261(5127):1411-1418.

Ellington AD. 1994. Aptamers achieve the desired recognition. Curr Biol. 4(5):427-429.

Johnston WK, Unrau PJ, Lawrence MS, Glasner MR, Bartel DP. 2001. RNA-Catalyzed RNA Polymerization: Accurate and General RNA-Templated Primer Extension. Science. 292(5520):1319-1325.

Lincoln TA, Joyce GF. 2009. Self-Sustained Replication of an RNA Enzyme. Science. 323(5918):1229-1232.

Paul N, Joyce GF. 2002. A self-replicating ligase ribozyme. P Natl Acad Sci USA. 99(20):12733-12740

## Role of RNA in Biology

RNA, in one form or another, touches nearly everything in a cell. RNA carries out a broad range of functions, from translating genetic information into the molecular machines and structures of the cell to regulating the activity of genes during development, cellular differentiation, and changing environments.

RNA is a unique polymer. Like DNA, it can bind with great specificity to either DNA or another RNA through complementary base pairing. It can also bind specific proteins or small molecules, and, remarkably, RNA can catalyze chemical reactions, including joining amino acids to make proteins.

All the RNA in cells are themselves copies of DNA sequences contained in the genes of a cell's chromosomes. Genes that are copied—"transcribed"—into the instructions for making individual proteins are often referred to as "coding genes." The genes that produce RNAs used for other purposes are therefore called "noncoding RNA" genes.

### RNA molecules assemble proteins and modify other RNAs

Several key classes of RNA molecules help convert the information contained in the cell's DNA into functional gene products like proteins. Messenger RNAs (mRNAs) are copies of individual protein-coding genes, and serve as an amplified read-out of each gene's nucleic acid sequence. Two key noncoding RNAs participate in the assembly of the proteins specified by mRNAs. Ribosomal RNA (rRNA) constitutes the core structural and enzymatic framework of the ribosome, the machine that synthesizes proteins according to the instructions contained in the sequence of an mRNA. Transfer RNAs (tRNAs) use complementary base pairing to decode the three-letter "words" in the mRNA, each corresponding to an amino acid to be sequentially incorporated into a growing protein chain.

Most RNA molecules, once transcribed from the chromosomal DNA, require structural or chemical modifications before they can function. In eukaryotic cells, mRNAs are assembled from longer RNA transcripts by the spliceosome, which consists of spliceosomal RNAs and protein partners. Spliceosomal RNAs help discard intervening sequences (introns) from pre-mRNA transcripts and splice together the mRNA segments (exons) to create what can be a complex assortment of distinct protein-coding mRNAs from a single gene. Many noncoding RNAs also require post-transcriptional modifications. For instance, ribosomal RNAs receive numerous chemical modifications that are required for proper ribosome assembly and function. These modifications are introduced by protein enzymes in conjunction with specialized noncoding RNAs (called snoRNAs) that base pair with the rRNA and guide the modifying enzymes to precise locations on the rRNA.

Some RNAs possess intrinsic enzymatic activity and can directly catalyze RNA modification reactions. These catalytic RNAs include certain self-splicing RNA transcripts, ribozymes, and RNAse P, an RNA enzyme that trims the ends of tRNA precursors in essentially all cells.

### RNA molecules regulate gene expression

Regulation of the production of proteins from coding genes is the basis for much of cellular and organismal structure, differentiation, and physiology. Diverse classes of noncoding RNAs participate in gene regulation at many levels, affecting the production, stability, or translation of specific mRNA gene products.

In prokaryotes (for example, bacteria), small antisense RNAs exert a variety of gene regulatory activities by base pairing specifically to their target mRNAs. Also common in prokaryotes are riboswitches, noncoding RNA sequences that usually function as regulatory domains contained within longer mRNAs. Riboswitches regulate the activity of their host mRNAs by binding to small molecules such as nucleotides or amino acids, sensing the abundance of those small molecules and regulating the genes that make or use them accordingly.

Eukaryotic cells contain thousands of small RNAs associated with various RNA interference (RNAi) pathways. For example, microRNAs (miRNAs) are regulatory RNAs approximately 22 nt long that are produced from longer transcripts that contain a certain kind of double-stranded "hairpin" structure. miRNAs associate with a protein of the Argonaute class, and base-pair specifically to mRNAs to inhibit their stability or translation. There are hundreds of miRNA genes in plants and animals, and each miRNA can regulate the activity of hundreds of protein-coding genes. Therefore, miRNAs individually and collectively have a profound impact on the development and physiology of multicellular eukaryotes.

Small interfering RNAs (siRNAs) are similar in length to microRNAs and are also associated with Argonaute proteins. Unlike miRNAs, which are produced from specific genetic loci that have evolved to regulate mRNAs, siRNAs can derive from essentially any transcribed region of the genome. siRNAs typically act directly upon the locus from which they are produced. So, siRNAs occur in cells where genes are under ongoing self-regulation by RNAi.
A major role for certain classes of small noncoding RNAs is defense of the cell against viruses, transposons, and other nucleic acid sequences that pose a potential threat to cellular homeostasis or genome stability. The response of some cells against viral infection includes the production of siRNAs complementary to the virus. Many endogenous siRNAs in eukaryotic cells specify the silencing of transposons and repeat sequences that are already resident in the genome. Similarly, in animals the Piwi-associated RNAs (piRNAs) promote genome integrity by silencing transposons and repeat sequences.

Another class of regulatory RNA consists of diverse kinds of longer noncoding transcripts that generally function to regulate the expression of distant genetic loci, often by suppressing or promoting their transcription. For example, the rox RNAs of the fruit fly seems to facilitate the remodeling of chromosome structure to allow the male X chromosome to be transcribed at twice the rate as a single X chromosome in females, which have two X's. Similarly, the Xist RNA in mammals helps inactivate one of the two X chromosomes in females, allowing males and females to have equivalent levels of gene expression from the X chromosome. Xist is one example of a broader class of very versatile regulatory RNAs known as long intergenic noncoding RNAs (lincRNAs). lincRNAs can act as scaffolds for the assembly of complexes of transcriptional regulatory proteins, and can facilitate the recruitment of defined combinations of protein regulators to specific genes.

This is an official Page of the University of Massachusetts Medical School

RNA Therapeutics Institute (RTI) &bull 368 Plantation St Worcester, Massachusetts 01605

Preface
I RNA as an Enzyme
1 Cleavage of RNA by RNAse P from Escherichia coli
I. Introduction
II. Aspects of the RNase P Reaction
III. Studies of Enzyme-Substrate Interactions
IV. Structure-Function Relationships in Ml RNA
V. Studies of the Protein Subunit of RNase P
VI. Hybrid Enzymes
VII. Conclusion
References
2 Bacillus Subtilis RNase
I. The RNase P Components
II. Mechanism of RNase P Cleavage
III. Toward the Higher-Order Structure of RNase P RNA
IV. Structure-Function Relationships in the RNase P RNA
V. Why Is the Catalytic Element of RNase P Composed of RNA?
References
3 Multiple Enzymatic Activities of an Intervening Sequence RNA from Tetrahymena
I. Introduction
II. Self-Splicing RNA
III. The IVS RNA Enzyme
References
4 Processing and Genetic Characterization of Self-Splicing RNAs of Bacteriophage
I. Introduction
II. Group I Splicing Mechanism for T4 td RN
III. Nondirected Mutagenesis and Delineation of Two Functional Domains for Splicing in the td Intron
IV. Multiple Self-Splicing Introns in T4
V. Conclusions
References
II RNA Splicing
5 The Mammalian Pre-Messenger RNA Splicing Apparatus: A Ribosome in Pieces?
I. Introduction
II. The Discovery of snRNPs
III. The snRNPs-and-Splicing Hypothesis
IV. snRNP Components and Structure
V. Is Eukaryotic RNase P an Sm snRNP?
VI. U1 snRNPs Bind 5' Splice Sites
VII. U2, U5, and U4/U6 snRNPs Also Participate in Splicing
VIII. The Spliceosome-Ribosome Analogy
References
6 Exon Sequences and Splice Site Proximity Play a Role in Splice Site Selection
I. Introduction
II. Exon Sequences and Splice Site Proximity Play a Role in Splice Site Selection
III. The Pattern of Splice Site Selection Is Altered in Different Extract Preparations and in Diluted Extracts
IV. Splice Site Selection Can Be Altered by Competition in Trans
V. Discussion
References
7 Factors That Influence Alternative Splice Site Selection in Vitro
I. Introduction
II. Materials and Methods
III. Results
IV. Discussion
References
8 Messenger RNA Splicing in Yeast
I. An Overview of Nuclear mRNA Splicing
II. Preliminary in Vitro and in Vivo Characterization of Yeast mRNA Splicing
III. Characterization of Mutations in the Splicing Process
IV. The RNA Gene Products and the Spliceosome
V. Speculations
References
9 Architecture of Fungal Introns: Implications for Spliceosome Assembly
I. Introduction
II. Branch Site-3' Splice Junction Relationship
III. Branch Site-5' Splice Junction Relationship
IV. Perspectives
References
10 RNA Joining and Trypanosome Gene Expression
I. Introduction
II. Materials and Methods
III. Results
IV. Discussion
V. Summary
References
III RNA Viruses
11 The Polio virus Genome: A Unique RNA in Structure, Gene Organization, and Replication
I. Introduction
II. Translation and Processing of the Polyprotein
III. RNA Replication
IV. Conclusions
References
12 Permanent Expression of Influenza Virus Genes Coding for Transcriptase Complexes: Complementation of Viral Mutants
I. Establishment of a Functional Expression System
II. Addition of Nuclear Protein to the Transcription Complex
III. Conclusions and Outlook
References
13 Molecular Mechanisms of Pathogenesis
Text
References
IV RNA in DNA Replication
14 Changes in RNA Secondary Structure May Mediate the Regulation of IncFII Plasmid Gene Expression and DNA Replication
I. Introduction
II. RNA Secondary Structure Predictions
III. Discussion
References
15 Regulation of Co IE 1 DNA Replication by Antisense RNA
I. Introduction
II. RNA Primer Formation
III. Regulation of Primer Formation
IV. Binding of RNA I to RNA II
V. Importance of the Rate of Binding of RNA I to RNA II
VI. Secondary Structure of RNA II and Its Alteration by Binding of RNA I
VII. Conclusions
References
16 A Transfer RNA Implicated in DNA Replication
I. Introduction
II. Expression of the DNA Y Gene
III. Implication in DNA Replication
IV. Summary and Models for Replication Role
References
V RNA: Structure, Function, and Isolation
17 Stable Branched RNA Covalently Linked to the 5' End of a Single-Stranded DNA of Myxobacteria
I. Introduction
II. DNA Structure of Stigmatella aurantiaca msDNA
III. RNA Sequence of RNA-Linked msDNA
IV. Determination of the Linkage between RNA and msDNA
V. Gene Arrangement of the Coding Regions for msDNA (msd) and msdRNA (msr) on the Chromosome
VI. Biosynthesis of Branched RNA-Linked msDNA
VII. How Is Branched RNA-Linked msDNA Synthesized?
References
18 Recognition of RNA by Proteins
I. A Simple RNA-Protein Interaction
II. A Complex RNA-Protein Interaction
References
19 A New Role for Transfer RNA: A Chloroplast Transfer RNA Is a Cofactor in the Conversion of Glutamate to Delta-Aminolevulinic Acid
I. Introduction
II. RNA DALA Is a Unique Glutamine-Accepting tRNA
III. RNA as a Cofactor: Possible Reaction Mechanism
IV. Outlook
References
20 Natural Suppressor Transfer RNA in Eukaryotes: Its Implication in the Evolution of the Genetic Code and Expression of Specific Genes
I. Introduction
II. Isolation from Tetrahymena of the tRNA Gene and tRNA Corresponding to the Termination Codon UAA
III. Deviation of the Genetic Code of Tetrahymena from the Universal Genetic Code
IV. Evolution of Glutamine tRNAs Recognizing UAA and UAG Termination Codons in Tetrahymena
V. Isolation of a Natural UAG Suppressor Glutamine tRNA from Mouse Cells
VI. A Large Increase of tRNAg[UG in Mouse Cells Infected with Mo-MuLV
VII. Concluding Remarks
References
21 The Purification of Small RNAs by High-Performance Liquid Chromatography
I. Introduction
II. Materials and Methods
III. Results
IV. Discussion
References
22 Comparative Studies on the Secondary Structure of the RNAs of Related RNA Coliphages
I. Introduction
II. Results
III. Discussion
References
VI RNA in Regulation and Repression
23 Autogenous Regulation of Transcription of the crp Operon by a Divergent RNA Transcript
I. Introduction
II. Activation of a Divergent Promoter by Cyclic AMP-CRP is Required for crp Autoregulation
III. Autoregulation of crp Is Mediated by Divergent RNA
IV. A Model for crp Autoregulation
V. Conclusion
References
24 The Role of Translational Regulation in Growth Rate-Dependent and Stringent Control of the Synthesis of Ribosomal Proteins in Escherichia Coli
I. Secondary Structure of the LI Target Site on L11 mRNA
II. Growth Rate-Dependent Control of Ribosomal Protein Synthesis
III. Stringent Control of Ribosomal Protein Synthesis
IV. Concluding Remarks
References
25 Sequence and Structural Elements Associated with the Degradation of Apolipoprotein II Messenger RNA
I. Introduction
II. Effect of 5' Noncoding Sequences on apo II mRNA Stability
III. Use of Enzymatic Probes and Reverse Transcriptase to Analyze apo II mRNA Secondary Structure
IV. Analysis of apo II mRNA in Polysomes
V. Summary and Perspective
References
26 A New Immune System against Viral Infection Using Antisense RNA: micRNA-Immune System
I. Introduction
II. micRNA Mutagenesis
III. micRNA-Immune System
IV. Conclusion
References
27 Regulation of IS10 Transposase Expression by RNA/RNA Pairing
I. Introduction
II. Biological Role of Multicopy Inhibition
III. Molecular Mechanism of Multicopy Inhibition
IV. Mechanism of Pairing between RNA-IN and RNA-OUT In Vitro
V. In Vivo Phenotypes of Mutations in RNA-OUT
References
28 Characterization and Functional Analysis of the Factors Required for Transcription of the Adenovirus Major Late Promoter
I. Introduction
II. Fractionation and Functional Analysis of the Factors Required for Transcription of the Adenovirus Major Late Promoter
III. Concluding Remarks
References
Index

## Seeking Proof With Peptoids

Of course, the key to all this lies in actual experimentation. “Everything that goes back further than 2.5 to 3 billion years is speculation,” said Erich Bornberg-Bauer, a professor of molecular evolution at the Westfälische Wilhelms University of Münster in Germany. He described Dill’s work as “really a proof of concept.” The model still needs to be tested against other theoretical models and experimental research in the lab if it is truly to put up a good fight against the RNA world hypothesis. Otherwise, “it’s like the joke about physicists [assuming] cows are perfectly elastic spherical objects,” said Andrei Lupas, director of the department of protein evolution at the Max Planck Institute for Developmental Biology in Germany, who believes in an RNA-peptide world, in which the two coevolved. “Any significance ultimately comes from empirical approaches.”

That’s why Zuckermann, one of Dill’s co-authors on the PNAS paper, has begun working on a project that he hopes will confirm Dill’s hypothesis.

Twenty-five years ago, around the time that Dill proposed his HP protein-folding model, Zuckermann was developing a synthetic method to create artificial polymers called peptoids. He has used those nonbiological molecules to create protein-mimicking materials. Now he’s using peptoids to test the HP model’s predictions by examining how sequences fold and whether they would make good catalysts. In the course of this experiment, Zuckermann said, he and his colleagues will be testing thousands of sequences.

That’s sure to be messy and difficult. Dill’s HP model is highly simplified and doesn’t account for many of the complicated molecular details and chemical interactions that characterize real life. “This means we will run into atomic-level realities that the model is not capable of seeing,” Zuckermann said.

One such reality might be that a pair of foldamers would aggregate instead of catalyzing each other’s production. Skeptics of Dill’s hypothesis worry that it would be far easier for the hydrophobic patches to interact with one another instead of with other polymer chains. But according to Pohorille, the potential for aggregation doesn’t automatically mean Dill is wrong about needing those hydrophobic patches to get autocatalysis started. “Modern enzymes aren’t just smooth balls. Enzymes contain crevices that assist the process of catalysis,” he explained. If there’s aggregation between the foldamers through their landing pads, it’s possible that the resulting structure could possess such features, too.

“Even if it seems unlikely, science has to consider all the hypotheses,” Bornberg-Bauer added. “That’s what Dill is doing.”

“This model gives experimentalists like me a starting point,” Zuckermann said. “It lays down the challenge to find these primitive catalysts, to show how they work, to say: This could have really happened.”

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## ›RNA as an enzyme‹

↗ RNA by itself is catalytically active: group I, group II introns, RNase P, and hammerhead.

↗ RNA as mobile genetic elements: excised introns can reinsert into the genome.

↗ Spliceosomes are large RNA-protein complexes mediating nuclear pre-mRNA splicing.

↗ Alternative pre-mRNA splicing generates multiple proteins from a single gene.

↗ RNA as a catalytic enzyme and as a genetic element led to the “RNA world hypothesis.”

## Key Concepts and Summary

• During transcription, the information encoded in DNA is used to make RNA.
• RNA polymerase synthesizes RNA, using the antisense strand of the DNA as template by adding complementary RNA nucleotides to the 3&rsquo end of the growing strand.
• RNA polymerase binds to DNA at a sequence called a promoter during the initiation of transcription.
• Genes encoding proteins of related functions are frequently transcribed under the control of a single promoter in prokaryotes, resulting in the formation of a polycistronic mRNA molecule that encodes multiple polypeptides.
• Unlike DNA polymerase, RNA polymerase does not require a 3&rsquo-OH group to add nucleotides, so a primer is not needed during initiation.
• Termination of transcription in bacteria occurs when the RNA polymerase encounters specific DNA sequences that lead to stalling of the polymerase. This results in release of RNA polymerase from the DNA template strand, freeing the RNA transcript.
• Eukaryotes have three different RNA polymerases. Eukaryotes also have monocistronic mRNA, each encoding only a single polypeptide.
• Eukaryotic primary transcripts are processed in several ways, including the addition of a 5&rsquo cap and a 3&prime-poly-A tail, as well as splicing, to generate a mature mRNA molecule that can be transported out of the nucleus and that is protected from degradation.

## 5.3 Protein Synthesis Requires RNA

DNA is not the only nucleic acid involved in expressing the message in your genes. Ribonucleic acid (RNA) is another large molecule that is involved in protein synthesis. RNA is similar to DNA in that it also consists of a series of nucleotides anchored by a backbone of repeating sugars and phosphates.

RNA differs from DNA in that it is typically single-stranded, rather than double-stranded. Also, the ribose sugar in RNA’s sugar-phosphate backbone is different from the deoxyribose in DNA. And although three of the four nitrogenous bases of RNA are the same as DNA (adenine (A), guanine (G), cytosine (C)), RNA has the base uracil (U) instead of thymine (T).

Figure 5.3 A comparison of DNA and RNA.

#### There are three types of RNA involved in protein synthesis

RNA is more diverse than DNA, occurring in several different functional types, three of which are essential to making proteins:

1. mRNA, or messenger RNA, conveys DNA’s genetic message in the form of an RNA transcript. An RNA transcript is a sequence of RNA bases complementary to the DNA bases in a gene that is being expressed through protein synthesis.
2. tRNA, or transfer RNA, aligns the mRNA transcript with amino acids, the fundamental building blocks of a protein. Each amino acid has a corresponding tRNA molecule, itself complementary to bases in mRNA.
3. rRNA, or ribosomal RNA, comprises much of the ribosome, a two-unit cellular structure that is the site of protein synthesis. Typically, human cells each contain several million ribosomes.

## Glycans May Bind to RNA, Initial Findings Suggest

##### Emily Makowski
###### Oct 7, 2019

F or the first time, scientists have found that complex sugars called glycans may bind to some RNA molecules, according to a bioRxiv preprint published September 30. The findings could substantially alter the current perception of RNA’s function.

“There really is no framework in biology as we know it today that would explain how RNA and glycans could ever be in the same place at the same time, much less be connected to each other,” senior author Carolyn Bertozzi, a chemical biologist at Stanford University, tells The Scientist.

Bertozzi’s lab found the sugars attached to RNA while studying glycosylation, a reaction where sugar molecules are attached to proteins or other organic molecules, in a human cell line. Glycosylation has many functions, including helping proteins fold and cells adhere to one another, and is the mechanism behind different blood types. First author Ryan Flynn, a postdoc in Bertozzi’s lab, was trying to label glycoproteins when he spotted what seemed to be a glycan attached to RNA, a surprising finding that the researchers had never seen before. Further investigations determined that sugars called N-linked glycans were sticking to a subset of noncoding RNA molecules including small Y RNAs, which may have a role in DNA replication.

“It was a really weird discovery. At first we were skeptical. . . We tried to shoot it down in every way that we could think of, and it just kept holding up,” says Bertozzi.

They tried to separate out any proteins from the sample, but after a variety of treatments found that the sample was only sensitive to enzymes that cut up RNA. “We were kind of left with the answer that it was RNA,” Flynn tells The Scientist. Further research suggested that this RNA, which the researchers have termed glycoRNA, is also found in mouse and hamster cell cultures and in cells that were taken from living mice.

### See “The RNA Age: A Primer”

The researchers don’t yet know how the RNA and the sugars are bound, because it was impossible to separate the two biopolymers except by using enzymes to digest either the RNA or the sugar. They believe the RNA and glycan “are somehow connected together through some linkage that is not a protein, or at least not a protein that’s recognized by a protease,” says Bertozzi, adding that the connection could be formed directly by covalent bonds.

RNA is normally found only in the nucleus and cytosol of cells, while glycosylation is thought to occur in the endoplasmic reticulum and Golgi bodies. For the two biopolymers to be found together, either RNA or glycans must enter one of these cell compartments in a way that was previously undetected, or there may be a molecule that acts as a go-between. “Whatever it is, it’s a completely unknown biology,” says Bertozzi.

Many of the glycoRNAs that were observed are known to contain RNAs that antibodies bind to in autoimmune conditions such as lupus. It is currently unknown why these RNAs might provoke an immune response.

Other RNA researchers expressed excitement about the paper and urged more research. “This paper, if verified, would certainly open up an entirely new direction of research investigating gene expression, gene regulation, quality control of transcription, and RNA turnover,” Richard Cummings, a professor of surgery at Harvard Medical School and the director of the National Center for Functional Glycomics who was not involved with the study, tells The Scientist.

“I was surprised and excited to see it. It’s an unexpected and thought-provoking observation,” says Torsten Krude at the University of Cambridge, a Y RNA researcher who was also not involved with the work. “If the results are consistent and verified by others, and if it holds the test of time and scrutiny, it would be an exciting new aspect to RNA biology.”

Bertozzi and Flynn have received feedback from other researchers since they announced the preprint on Twitter. “I think the more input we get, the better job we’ll be able to do to dig deeper and get to the bottom of this,” says Bertozzi.

## The RNA World Hypothesis

Thus, it's very likely that the first ribosomes were only made of RNA. The protein parts of the molecule were likely added later, and helped prevent it from breaking down. This is part of a wider body of work focused on the very beginning of life on Earth—the RNA World Hypothesis.

The hypothesis considers the observations that:

1. RNA can be used to store information in its sequence
2. RNA can perform functional roles and catalyze (help or speed up) reactions

It suggests that the first life forms used RNA for both these functions.

DNA and proteins evolved later and are more stable. But there was likely a time when all organisms had RNA genomes and cellular functions depended on RNA-based enzymes.