Maria Chiaffarano

BARTEL AND SZOSTAK EXPERIMENT

HOW DID LIFE BEGIN?

FOUR OVERLAPPING STAGES• Scientists used four stages to understand the origin of life

• Stage 1

• Nucleotides and amino acids were produced prior to the existence of cells

• Stage 2

• Nucleotides became polymerized to form RNA and/or DNA and amino acids become polymerized to form proteins

• Stage 3

• Polymers became enclosed in membranes

• Stage 4

• Polymers enclosed in membranes acquired cellular properties

CHEMICAL SELECTION

• What is chemical selection?

• Chemical within a mixture has special advantageous properties

• Properties allow it to increase in amount

• Hypothesis

• The cellular characteristics that exist today evolved from an “RNA world”

RNA WORLD

• Scientists believe that the world used to consist of RNA based organisms

• Studied the building blocks

• Amino acids and nucleotides

KEY FUNCTIONS OF RNA

• RNA has three key functions that encourage scientists to favor it as the first macromolecule found in protobionts

• Ability to store information in its nucleotides

• Nucleotide sequence has the capacity for self-replication

• RNA has many enzymatic functions

• Act as ribozymes

ADDITIONAL ADVANTAGES OF RNA

• DNA and proteins are not as versatile as RNA

• DNA has limited catalytic activity

• Proteins do not undergo self-replication

• However:

• RNA can perform functions that are characteristics of proteins while simultaneously serving as genetic material with replicative and informational functions

BARTEL AND SZOSTAK EXPERIMENT

• David Bartel and Jack Szostak

• First study that used RNA molecules with a particular function (1993)

• Synthesized a mixture of 10^15 RNA molecules also known as “long RNA”

• First region (5’ end) constant region among all “long RNAs” (identical)

• Second region, variable region (220 nucleotides)

• Hypothesized that the variable region could possibly result in long RNA with the enzymatic ability to catalyze a covalent bond between two adjacent nucleotides

MATERIALS

• Many copies of short RNA

• Had a tag sequence that binds tightly to “beads” (column packing material)

• Had a complementary sequence to a site in the constant region of the long RNA

• No variable region, all the same

• Long RNAs with the constant and variable regions

• Variable regions were made using a PCR step (caused mutations in the region)

OVERVIEW OF THE EXPERIMENT

STEP 1

• The long RNAs and short RNAs were incubated together

• Promote hydrogen bonding

• Time was given for the molecules to form covalent connections (only if the RNA had the enzymatic ability to form covalent bonds)

• The long RNA molecule variable regions may rarely have enzymatic ability to connect the 3’end of short RNA to 5’end of long RNA

STEP 2

• The mixture passed through a column of beads

• The beads would bind to the tag sequence of the short RNA only!

• Tag sequence promoted the binding of the short RNA to the beads

• IF the long RNA had the ability to bind to the short RNA, it would also be attached to the beads

• Additional liquid was added to filter out the long RNAs that did not covalently bond to the short RNAs

STEP 3

• The main purpose of this step was to get rid of the beads

• Low pH solution was added in order to prevent the tag sequence from binding to the beads

• The tightly bound RNAs are flushed out of the column

• They were flushed into a flask and labeled “Pool #1”

• Beads were left behind

STEP 4

• Pool #1 was used to make a second batch of long RNA molecules

• Polymerase chain reaction (PCR) was used

• Variable regions were derived from the variable regions of pool #1 RNA molecules, expected to have enzymatic activity

• Reverse transcriptase was used to make cDNA

• PCR primers recognized beginning and end of the long RNA sequence and copied only this region

• cDNA used as a template to make long RNA via RNA polymerase

STEPS 5 & 6

• The procedure was repeated in order to generate 10 consecutive pools of RNA molecules

• A sample of the original population (Pool #1) and each of the following 10 pools were collected in order to test for the enzymatic ability to catalyze a covalent bond between adjacent nucleotides

CONCLUSION

• Data:

• Chemical selection is possible

• Scientists knew this because the experiment showed that there was an increase in covalent bond formation from pool 1 to pool 10

• In each generation, the catalytic enzyme activity increased

• Pool #10’s enzymatic activity was approximately 3 million times higher than the original random pool of molecules