Segment 1 -Clay-Catalyzed RNA Polymerization Activity

Astrobiology

Clay-Catalyzed RNA Polymerization Activity

Level: Grades 9 - 12

Overview

This lesson was developed for use in general biology, chemistry, or geology classrooms to teach current Origin of Life theories, especially the feasibility of the "RNA world" hypothesis (see readings). As a biology teacher I always find teaching about "the origin of life on earth" fascinating but at the same time frustrating. The information given in most textbooks is limited at best, although more is being uncovered in this field daily. While no one is ever likely to know the exact mechanism that led to life on early earth, the notion that RNA played a pivotal role is gaining momentum. Origins research is currently a rich area of speculation and investigation. The nature of the question has captured the imagination of many and has become part of a developing field of research called Astrobiology.

A central problem with teaching about the origins of life on earth lies in explaining how the jump from a collection of organic molecules to even primitive life forms could have come about. With recent work on RNA and ribozymes, many researchers think they are moving closer to solving this mystery. The RNA world hypothesis proposes that RNA was the first molecule of biological significance, acting as an enzyme-like catalyst (ribozyme) as well as a self-replicating information storage molecule. Many students are already familiar with RNA and its various cellular functions, such as its role in protein synthesis. This is a lesson showing how short RNA fragments could have been synthesized in the presence of clay catalysts in a prebiotic environment and hints at how life could have emerged.

This work was supported by Dr. James Ferris, Director of the New York Center for Studies on the Origins of Life and Professor of Chemistry at Rensselaer Polytechnic Institute in Troy, New York. Dr. Ferris and co-workers have demonstrated, among other things, polymerization of activated RNA nucleotides in the presence of a clay catalyst as described in this lesson. I was fortunate to be able to spend time in Dr. Ferris' lab at Rensselaer during the summer of 2000 along with my student Liz Vrolyk.

Support Materials

Readings: Included in this module are two readings:

"From Building Blocks to the Polymers of Life" provides a general overview of the Origins of Life theories and reviews current research in the field, edited for student use.
"Clay and the Origins of Life" provides background information on clay structures and the possible role of minerals in the formation of RNA polymers on early earth, edited for student use.

Copies of both of these readings can be made available to students, or can be used as background information for the teacher. Additional teacher notes are provided below.

Teacher Notes

The geochemistry of clays is an important part of this lesson because clay is acting as the catalyst in the formation of short RNA oligonucleotides. No catalysts of the type present in life today (enzymes, ribozymes) would have been present in a prebiotic environment, but other minerals besides clay may have catalyzed other reactions leading to the origins of life. Clay structures vary widely and could be the subject of a course unto themselves. What follows is the essential geochemical information one would need to proceed with this lesson. Geology references are included.

Clay Structure

Clay minerals are a major product of weathering processes. Complete weathering of volcanic igneous rocks, especially feldspars, leads to clay mineral formation. Clay minerals never occur as large crystals but are usually found in what are known as "muds"; the predominant clay particles are often less than one micrometer (<10^-4 cm) in diameter, placing them in the colloidal size range. Because of their small particle size, clays have large surface area to mass ratios. Clays are classified on the basis of their structures.

The following explanation and diagrams of clay structures are adapted from Physical Geology by Plummer, McGeary and Carlson.⁸

The two most abundant elements of earth's crust, silicon and oxygen, combine to form the basic building blocks for most common minerals, including clays. Silica is a term for oxygen combined with silicon. Silicates are substances that contain silica. Most silicate minerals also contain one or more other elements such as aluminum.

General silicates are made of "building blocks" of four oxygen atoms packed together around a single, much smaller, silicon atom. The four-sided, pyramidal geometric shape is called a tetrahedron. A single silicon-oxygen tetrahedron is a complex ion with a formula of SiO₄^-4 because silicon has a charge of +4 and the four oxygen ions have 8 negative charges (-2 for each oxygen atom). For the silicon-oxygen tetrahedron to be stable within a crystal structure, it must either be balanced by enough positively charged ions or share oxygen atoms with adjacent tetrahedrons and therefore reduce the need for extra, positively charged ions. There is a variety of arrangements possible for silicates (depending on how many oxygen atoms are shared with other tetrahedrons) which range from isolated silicate structures (single tetrahedrons), to single and double chain structures, to sheet silicates (clays are in this group) and finally framework silicates such as quartz.

When each tetrahedron shares three oxygen ions, the result is a sheet silicate structure, characteristic of the mica group and the clay group of minerals. Octahedral sheet structures also occur, especially with Al⁺³ in coordination with two oxygen atoms and an OH ^- group. Clays can include octahedral sheets as well as tetrahedral sheets. There are various substitution possibilities within clays. Sometimes aluminum can substitute for silicon in the silicate tetrahedron, for example.

The clay that is used as a catalyst in the RNA polymerization reaction described in this lesson is montmorillonite, which is referred to as an aluminosilicate clay. It consists of three "layers": an aluminum-containing octahedron layer sandwiched between two silicate layers. The O^2- at the apexes of the silica tetrahedra replaces the OH^- groups of the octahedral layer, and are shared between the layers. This three-layer grouping is referred to as a platelet. The platelets stack in various ways. Water is absorbed between the platelets. There is an unsatisfied negative charge on the face of the clay platelet caused by structural substitutions or vacancies in the octahedral and/or tetrahedral layers. For example Mg²⁺ or another divalent cation may substitute for Al³⁺ in the octahedral layer, or Al³⁺ or Fe³⁺ may replace Si⁴⁺ in the tetrahedral layer.

Cations such as Ca²⁺, Mg²⁺, and Na⁺ are attracted to the spaces between the platelets due to the net negative charge on the "faces" of the platelets. These cations can be exchanged readily through washing.

Montmorillonite is also classified as an expansive, or swelling, clay. If water is added to the montmorillonite, the water molecules are absorbed into the spaces between the clay platelets, especially if the associated cation is Na⁺. This result is a large increase in volume, sometimes up to several hundred percent. The pressure generated can be up to 50,000 kilograms per square meter: sufficient to lift a good-sized building. If a building is erected on expansive clay that subsequently gets wet, a portion of the building will be shoved upward. In all likelihood the building will break. Some people think that expansive soils have caused more damage than earthquakes and landslides combined. In addition, absorption of organic molecules is often associated with an expansion of the layers. Chinese potters actually combined animal urine with kaolinite clay to increase the swelling of the clay.

The chemical structures of clays indicate that they could have served as concentrators for specific populations of molecules as well as providing metal ions for catalysts. We know that clay minerals have surfaces that interact with organic molecules. It is this property that makes them good candidates for prebiotic synthesis of RNA oliognucleotides. Hydrophobic interactions take place between the purine bases of the nucleotides and the silicate surfaces of the clay. Divalent cations, associated with the negative phosphate groups of the nucleotides, bind the nucleotides to the negative surfaces of the clay platelets, positioning the nucleotides in such a way as to promote bond formation between the phosphate of one and the ribose sugar of an adjacent nucleotide. RNA formation occurs on the faces of the platelets. The exact mechanism is not known. There is no evidence for biopolymer formation in the same reaction performed in water.

Students

Instructions for the Activity: Blackline masters of RNA nucleotides and clay structures are provided. These could be enlarged for classroom use or used to make overhead transparencies.

Students should have an understanding of basic chemistry. It is also assumed that students have prior understanding of the structure and function of RNA, DNA, protein synthesis and enzyme function. This activity reinforces this information, but it is not intended to be used as a first exposure to these concepts. A logical sequence or timeline for presentation of material in this activity is as follows:

Start with a discussion of clay structure. Use the diagram of silica clay structure to familiarize the students with crystal structure. Depending on the background of your students and the focus of the course, this could include growing crystals, building crystals from atomic modeling kits, searching the Internet for crystal sites, etc. There are many crystal-related activities available from earth science and geology sources. If modeling kits are available, the students could build the aluminosilicate tetrahedrons and octahedrons and show how they stack.
Introduce or review the structure of RNA nucleotides. Biology students should be able to identify the three parts of a nucleotide: the ribose sugar, the phosphate group, and the base (A, C, U, or G). Emphasize the negative charge on the phosphate group. Research indicates that synthesis of the nitrogenous bases and ribose on early earth is within reason. Precursors could also have arrived on early earth on meteorites. A discussion of these points can be found in many of the references.

A more thorny problem is how the RNA nucleotides on early earth would have become "activated". In present day RNA synthesis, the RNA nucleosides (the sugar and the base unit) are bonded to a triphosphate group. As each monomer joins the growing end of a RNA strand, it loses two phosphate groups. Hydrolysis of the phosphate is the exergonic reaction that drives the polymerization of nucleotides to form RNA. RNA polymerization experiments done in Dr. Ferris' lab in the presence of a clay catalyst also use activated RNA mononucleotides (specifically phosphorimidazolides of RNA nucleosides, see references). How this or any other activated RNA nucleotide could have been formed on early earth is under investigation. We assume for this lesson that it is possible.

Here are Some Ideas for How to Use This Information

Purchase modeling clay at an art supply or a ceramics supply source. Possibly the art department at your school uses it. The terra cotta or buff variety is probably closer to what naturally occurring montmorillonite looks like. Ask the students to slide it between their fingers; to "play" with it. When handling a clay slurry, the "slimy" feel reflects the sliding of the aluminosilicate sheets over one another, analogous to the slippery nature of graphite.

Experiments could be devised to check how much water is absorbed by dry clay. First the clay needs to be spread out to dehydrate. It will crumble when dehydrated (if it is the kind that is used to do pottery). Also, the absorption of water can be compared to the absorption of a solution of water and organic compounds, such as urea. As a simple classroom activity, one could mix different concentrations of nucleotide and magnesium chloride with montmorillonite clay to observe the swelling; alternatively one could just mix a clay mineral with an organic compound (such as urea) that is known to absorb efficiently.¹⁰

If there is a source of clay in your community, students could take a field trip to dig their own clay to bring back to the classroom.

Students could model small tetrahedrons from the clay. Explorations of how these tetrahedrons can share "corners" could be done by joining the tetrahedrons in arrangements that occur in nature. (See silicate diagrams.) A study of different kinds of minerals, which exhibit each arrangement, could follow. Copy the diagram of clay crystal structure and have the students color in the tetrahedrons, octahedrons or individual ions. See geology references.

Copy the handout with the clay platelet and nucleotide diagrams. Explain to the students that this is a cross-sectional view of the platelet. Notice the surface of the platelet and associated negative charge. If clay is available have the students shape a clay rectangle that matches the dimensions on the diagram and then place the real clay on the diagram.

Use a hole-punch and different colors of paper to make an assortment of small discs, which will represent positive ions. The students can make a key for the ions. Make sure the Mg⁺⁺ ion is represented. Scatter these discs over the clay, where they will "stick".

Nucleotide diagrams are provided on the same page as the clay platelet. It is assumed that the students already have knowledge of nucleotide structure. For this activity it is important to stress the negative charge on the phosphate group. In an earlier exercise (not provided) students could make their own RNA nucleotides from the three components: the sugar, the phosphate and the base. Depending on the level of the students, define the 3' and 5' carbons contained in the ribose. Present-day RNA polymers have 3', 5' -phosphodiester bridges catalyzed by the enzyme RNA polymerase. Mononucleotide units are added to the 3'-hydroxyl ends of the RNA chain and thus RNA is formed in the 5' to 3' direction. This is a great place to review DNA and RNA synthesis with advanced students. This simulation provides ribonucleoside 5'-monophosphates. As discussed earlier, it is unclear what the activating group might have been for early ribonucleosides. We leave this to your speculation!

For the purpose of this activity generic ribonucleotides are provided. Ask students to trim around these structures so they are separate. The students can now position these cutouts on the paper clay platelets. The idea is to position them so that the charges are satisfied. The negative face of the platelet will attract positive ions, and in this case, Mg⁺² is the ion of choice. It has two positive charges so it can bind simultaneously to both the clay platelet face and the phosphate group on the nucleotide. The students should be able to arrange the ions and nucleotides on the faces of the clay platelet to satisfy the charges. In an additional activity with 9th graders I assigned some students the role of the magnesium ion and others were the nucleotides. We used their desks as the clay platelets and we did a role-play to show the action of the magnesium ions "sandwiched" between the nucelotides and the desk. The "magnesium ions" had two hands, one to grab the nucleotide and one to grab the clay platelet. When many "nucleotides" were lined up close together they joined hands to show polymerization. It worked!

With the paper simulation, when enough nucleotides are lined up in the right orientation, the phosphodiester bridge can be formed between the 3' hydroxyl and the 5' - monophosphate. In your simulation, this "bond" can be accomplished with tape. Notice that the RNA polymer continues to be bonded to the platelet face by the interaction of the Mg⁺⁺ ion attracting the phosphate group and the negative face of the clay. The surface attraction of a growing polymer increases the probability of elongation.

It has been shown that oliogomers of up to 10 to 15 nucleotides can form on Na⁺ montmorillonite in the presence of Mg⁺² at a pH of 8. RNA oliogomers of this length are not long enough to have significant catalytic activity (ribozymes). Experimental studies in which fresh activated monomer was added daily to a 10-mer primer over a 14-day period resulted in the incremental growth of the decamer to a mixture of 20-50 mers (Ferris et al. 1996).

For one last simulation, purchase a few packages of sugar wafer cookies, the kind that come in rectangular "platelets". These wafers, when viewed from the side, look analogous to the layers of a clay crystal. There are two triangular tetrahedral layers (the cookie part) with a layer of filling (the octahedral layer) in between them. Most of these wafers have a regular geometric "gridded" surface as well, much like a crystal. (OK, so the analogy is not perfect, but go with me here.) The wafers can be stacked to simulate clay platelets, with spaces in between the platelets for the RNA polymerization reaction to occur. Clay platelets have other stacking formations which could be explored using these wafers as models. Then you can eat them!

Bibliography

Brack, Andre. The Molecular Origins of Life, Assembling Pieces of the Puzzle. (Cambridge University Press; 1998) ISBN 0-521-56412-3
Brownlow, Arthur H. Geochemistry, Second Edition. (Prentice-Hall, Inc.; 1996) ISBN 0-13-398272-6
Ferris, J.P., Hill, A.R., Jr., Liu, R., and Orgel. L.E. 1996. Synthesis of long prebiotic oligomers on mineral surfaces. Nature 381:59-61
Fry, Iris. The Emergence of Life on Earth, A Historical and Scientific Overview. (Rutgers University Press; 2000) ISBN 0-8135-2740-6
Hay, Edward A. and McAlester, A. Lee. Physical Geology, Principles and Perspective, Second Edition. (Prentice-Hall, Inc; 1984) ISBN 0-13-669549-3
Langmuir, Donald. Aqueous Environmental Geochemistry. (Prentice Hall, NJ; 1997) ISBN 0-02-367412-1
Lahav, Noam. Biogenesis, Theories of Life's Origin. (Oxford University Press; 1999) ISBN 0-19-511755-7
Plummer, McGeary and Carlson, WCB. Physical Geology, Eighth Edition. (McGraw-Hill; 1999) ISBN 0-697-37404-1
Zubay, Geoffrey. Origins of Life on the Earth and in the Cosmos, Second Edition. (Harcourt Academic Press; 2000) ISBN 0-12-781910-X
William J. Hagan, Ph.D., Associate Professor of Chemistry, College of St. Rose, Albany, NY, personal communication

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Center for Studies of Origins of Life, Rensselaer Polytechnic Inst., Troy, NY 12180
http://www.origins.rpi.edu
Email: Origins of Life