NR 283 Unit 1 Discussion Topic – DNA & RNA
What is the structure of DNA? What is the structure of
... [Show More] RNA?
Answer
Identification of DNA as the Genetic Material
To fulfill its role, the genetic material must meet several criteria.
Information: It must contain the information necessary to make an entire organism. Transmission: It must be passed from parent to offspring.
Replication: It must be copied to be passed from parent to offspring.
Variation: It must be capable of changes to account for the known phenotypic variation in each species.
The data of many geneticists, including Mendel, were consistent with these four properties; however, the chemical nature of the genetic material cannot be identified solely by genetic crosses.
Experiments with Pneumococcus suggested that DNA is the genetic material.
Frederick Griffith Experiments with the bacterium Streptococcus pneumoniae.
Streptococcus pneumoniae comes in two strains.
S Smooth
Secrete a polysaccharide capsule (protects bacterium from the immune system of animals).
Produce smooth colonies on solid media.
R Rough
Unable to secrete a capsule.
Produce colonies with a rough appearance.
In addition, the capsules of two smooth strains can differ significantly in their chemical composition (see fig. 9.1).
Rare mutations can convert a smooth strain into a rough strain, and vice versa; however, mutations do not change the type of the strain.
In 1928, Griffith conducted experiments using two strains of Streptococcus pneumoniae: type IIIS and type IIR (see fig. 9.2).
Inject mouse with live type IIIS bacteria.
Mouse died.
Type IIIS bacteria recovered from the mouse’s blood.
Inject mouse with live type IIR bacteria.
Mouse survived.
No living bacteria isolated from the mouse’s blood.
Inject mouse with heat-killed type IIIS bacteria.
Mouse survived.
No living bacteria isolated from the mouse’s blood.
Inject mouse with live type IIR + heat-killed type IIIS cells.
Mouse died.
Type IIIS bacteria recovered from the mouse’s blood.
Griffith concluded that something from the dead type IIIS was transforming type IIR into type IIIS; he called this process transformation.
The substance that allowed this to happen was termed the transformation principle; Griffith did not know what it was.
The nature of the transforming principle was determined using experimental approaches that incorporated various biochemical techniques.
The Experiments of Avery, MacLeod and McCarty.
Avery, MacLeod and McCarty realized that Griffith’s observations could be used to identify the genetic material.
They did their experiments in the 1940s; at that time, it was known that DNA, RNA, proteins and carbohydrates are major constituents of living cells.
They prepared cell extracts from type IIIS cells containing each of these macromolecules.
Only the extract that contained purified DNA was able to convert type IIR into type IIIS.
Avery et al. conducted the following experiments (see fig. 9.3) to further verify that DNA, and not a contaminant (RNA or protein), is the genetic material.
Type IIR cells alone: No transformation.
Type IIR cells + Type IIIS DNA extract: Transformation.
Type IIR cells + Type IIIS DNA extract + DNase No transformation. Type IIR cells + Type IIIS DNA extract + RNase Transformation.
Type IIR cells + Type IIIS DNA extract + Protease Transformation.
Treatment of the DNA extract with RNase or protease did not eliminate transformation.
Treatment with DNase did.
Experiment 9A – Hershey and Chase provided evidence that the genetic material injected into the bacterial cytoplasm of Escherichia coli is bacteriophage T2 DNA.
In 1952, Alfred Hershey and Marsha Chase provided further evidence that DNA is the genetic material.
Hershey and Chase studied the bacteriophage T2; it is relatively simple because it is composed of only two macromolecules, DNA and protein (see fig. 9.4).
The Hershey and Chase experiment can be summarized as follows:
Used radioisotopes to distinguish DNA from proteins; 32P labels DNA specifically and 35S labels protein specifically.
Radioactively-labeled phages were used to infect non-radioactive Escherichia coli
cells.
After allowing sufficient time for infection to proceed, the residual phage particles were sheared off the cells and the phage ghosts were separated from the E. coli cells.
Radioactivity was monitored using a scintillation counter.
The hypothesis.
Only the genetic material of the phage is injected into the bacterium; isotope labeling will reveal if it is DNA or protein.
Testing the hypothesis (see fig. 9.6).
Grow bacterial cells; divide into two flasks.
To one flask add 35S-labeled phage; to the second flask add 32P-labeled phage. Allow infection to occur.
Agitate solutions in blenders to shear the empty phages off the bacterial cells. Centrifuge at 10,000 rpm.
The heavy bacterial cells sediment to the pellet while the lighter phages remain in the supernatant.
Count the amount of radioisotope in the supernatant with a scintillation counter and compare with the starting amount.
The data (see fig. 9.6).
In the sample containing 35S-labeled phage, agitation in the blender removes 80% of the 35S from the E. coli cells.
In the sample containing 32P-labeled phage, agitation in the blender removes only 35% of the 32P from the E. coli cells; most (65%) remains with intact E. coli cells
Interpreting the data.
These results suggest that DNA is injected into the bacterial cytoplasm during infection; this is the expected result if DNA is the genetic material.
RNA functions as the genetic material in some viruses.
In 1956, A. Gierer and G. Schramm isolated RNA from the tobacco mosaic virus (TMV), a plant virus.
Purified RNA caused the same lesions as intact TMV viruses; therefore, the viral genome is composed of RNA.
Since that time, many RNA viruses have been found (see table 9.1).
Nucleic Acid Structure
DNA and RNA are large macromolecules with several levels of complexity (see fig. 9.7).
Nucleotides form the repeating units. Nucleotides are linked to form a strand.
Two strands can interact to form a double helix.
The double helix folds, bends and interacts with proteins resulting in 3-D structures in the form of chromosomes.
Nucleotides are the building blocks of nucleic acids (see fig. 9.8).
The nucleotide is the repeating structural unit of DNA and RNA; it has three components.
A phosphate group. A pentose sugar.
A nitrogenous base.
In DNA, the sugar is deoxyribose and the bases are guanine, cytosine, adenine, and thymine. In RNA, the sugar is ribose and the bases are guanine, cytosine, adenine, and uracil.
Terminology of nucleic acid units (see fig. 9.10).
Base + sugar nucleoside Example
Adenine + ribose = Adenosine
Adenine + deoxyribose = Deoxyadenosine Base + sugar + phosphate(s) nucleotide
Example
Adenosine monophosphate (AMP) Adenosine diphosphate (ADP) Adenosine triphosphate (ATP)
Nucleotides are linked together to form a strand (see fig. 9.11).
Nucleotides are covalently linked together by phosphodiester bonds.
A phosphate connects the 5’ carbon of one nucleotide to the 3’ carbon of another; therefore, the strand has directionality 5’ to 3’.
The phosphates and sugar molecules form the backbone of the nucleic acid strand; the bases project from the backbone.
A few key events led to the discovery of the double-helix structure of DNA.
In 1953, James Watson and Francis Crick discovered the double helical structure of DNA.
The scientific framework for their breakthrough was provided by other scientists including:
Linus Pauling (see fig. 9.12).
In the early 1950s, Pauling proposed that regions of protein can fold into a secondary structure – an -helix.
To elucidate this structure, he built ball-and-stick models.
Rosalind Franklin (see fig. 9.13).
Working in the same laboratory as Maurice Wilkins, Franklin used X-ray diffraction to study wet fibers of DNA; the diffraction pattern she obtained suggested several structural features of DNA.
Helical
More than one strand
10 base pairs per complete turn Erwin Chargaff (see Experiment 9B below).
Chargaff pioneered many of the biochemical techniques for the isolation, purification and measurement of nucleic acids from living cells.
It was already known then that DNA contained the four bases: A, G, C and T.
Experiment 9B – Chargaff found that DNA has a biochemical composition in which the amount of adenine (A) equals the amount of thymine (T) and the amount of guanine (G) equals the amount of cytosine (C).
The hypothesis.
An analysis of the base composition of DNA in different species may reveal important features about the structure of DNA.
Testing the hypothesis (see fig. 9.14).
The following cells were used in Chargaff’s experiment: Escherichia coli, Streptococcus pneumoniae (type III), yeast, turtle red blood cells, salmon sperm cells, chicken red blood cells, and human liver cells.
For each cell type, chromosomal material, which contains both DNA and protein, was extracted.
Protein was removed.
The DNA was hydrolyzed to release the bases from the DNA strands. The bases were separated by paper chromatography.
The bands containing the bases were extracted and the amounts of each base were determined by spectroscopy.
Base content in the DNA from different organisms was compared.
The data (see fig. 9.14). Interpreting the data.
The compelling observation was that: Percent of adenine = percent of thymine. Percent of guanine = percent of cytosine.
This observation became known as Chargaff’s rule, which was crucial evidence that Watson and Crick used to elucidate the structure of DNA.
Watson and Crick deduced the double-helical structure of DNA.
Familiar with all of these key observations, Watson and Crick set out to determine the structure of DNA.
They tried to build ball-and-stick models that incorporated all known experimental observations.
A critical question was how the two (or more) strands would interact.
An early hypothesis, which was incorrect, proposed that the strands interact through phosphate-Mg++ crosslinks (see figure 9.15).
They went back to the ball-and-stick units and built models with the sugar-phosphate backbone on the outside, bases projecting toward each other.
They first considered a structure in which bases form H bonds with identical bases in the opposite strand; i.e., A to A, T to T, C to C, and G to G; model building revealed that this also was incorrect.
They then realized that the hydrogen bonding between A and T resembled that between C and G, so they built ball-and-stick models with AT and CG interactions; these were consistent with all known data about DNA structure (see figure 9.16).
Watson, Crick, and Maurice Wilkins were awarded the Nobel Prize in 1962; Rosalind Franklin died in 1958, and Nobel prizes are not awarded posthumously.
Molecular structure of the DNA double helix has several key features.
General structural features (see figures 9.17 and 9.18). Two strands are twisted together around a common axis. There are 10 bases and 3.4 nm per complete twist.
The two strands are antiparallel; one runs in the 5’ to 3’ direction and the other 3’ to 5’.
The helix is right-handed; as it spirals away from you, the helix turns in a clockwise direction. The double-bonded structure is stabilized by:
Hydrogen bonding between complementary bases.
A is bonded to T by two hydrogen bonds. C is bonded to G by three hydrogen bonds.
Base stacking.
Within the DNA, the bases are oriented so that the flattened regions are facing each other.
There are two asymmetrical grooves on the outside of the helix.
Major groove and minor groove.
Certain proteins can bind within these grooves; they can thus interact with a particular sequence of bases.
DNA can form a triple helix, called triplex DNA (see fig. 9.20).
In the late 1950s, Alexander Rich et al. discovered triplex DNA; it was formed in vitro using DNA pieces that were made synthetically.
In the 1980s, it was discovered that natural double-stranded DNA can join with a synthetic strand of DNA to form triplex DNA; the synthetic strand binds to the major groove of the naturally-occurring double-stranded DNA.
Triplex DNA formation is sequence specific; the pairing rules are – T binds to an AT pair in biological DNA and C binds to a GC pair in biological DNA.
Triplex DNA has been implicated in several cellular processes – replication, transcription, recombination – and cellular proteins that specifically recognize triplex DNA have been recently discovered.
As shown in fig. 9.20a, the synthetic DNA strand binds into the major groove according to specific base pairing rules; therefore, researchers can design a synthetic DNA to recognize the base sequence found in a particular gene.
When synthetic DNA binds to a gene, it inhibits transcription and can even cause mutations that inactivate the gene’s function; thus, synthetic DNA could be used to silence genes, perhaps those that have become overactive in cancer cells.
The three-dimensional structure of DNA within chromosomes requires additional folding and the association with proteins.
To fit within a living cell, the DNA double helix must be extensively compacted into a 3-D conformation; this is aided by DNA-binding proteins (see fig. 9.21).
This topic will be discussed in detail in the Chapter 10 lectures.
RNA molecules are composed of strands that fold into specific structures.
The primary structure of an RNA strand is much like that of a DNA strand (compare fig. 9.22 to fig. 9.11).
RNA strands are typically several hundred to several thousand nucleotides in length. In RNA synthesis, only one of the two strands of DNA is used as a template.
Although usually single-stranded, RNA molecules can form short double-stranded regions.
This secondary structure is due to complementary base-pairing of A to U and C to G, which allows short regions to form a double helix.
Different types of RNA secondary structures are possible (see fig. 9.23). Many factors contribute to the tertiary structure of RNA.
For example, base-pairing and base stacking within the RNA itself. Interactions with ions, small molecules and large proteins.
The tertiary structure of tRNAphe, the transfer RNA that carries phenylalanine, is shown in fig. 9.24.
Please see the Conceptual Summary and Experimental Summary for Chapter 9 on page 241.
This lecture outline was prepared from Genetics: Analysis and Principles, by Brooker, 2009 (3rd edition). It contains phrases and entire sentences taken verbatim from that source, and is in no way meant to represent original work by Mark Bierner. [Show Less]