Nucleic Acids Flashcards
Landmarks in DNA research
DNA as the genetic material
In the early 1950s it was still unclear wether genes were made of DNA or protein. Hershey and Chase used a virus, T2, that infects cells of the bacterium E. coli to investigate this. Viral proteins start being made in the cytoplasm of E. coli soon after T2 comes into contact with it, showing that the viral genes have entered the bacterium.
Viruses such as T2 consist only of DNA inside a protein coat. DNA contains phosphorus but not sulphur and protein contains sulphur but not phosphorus. Hershey and Chase used this difference to prepare two strains of T2, one having its DNA radioactively labelled with 32P and the over having its protein labelled with 35S.
These two strains of labelled T2 were each mixed with E. coli. After leaving enough time for the bacteria to be infected, the mixture was agitated in a high-speed mixer and the centrifuged at 10,000 pm to separate into a solid pellet containing the bacteria and liquid supernatant. A Geiger counter was used to locate the radioactivity.
Analysis of results
T2 binds to the surface of E. coli and injects its DNA into the bacterium. This explains the high proportion of radioactivity with the bacteria in the pellet when 32P was used. Agitation shakes many of the protein coats of the viruses off the outside of the bacteria and these coats remain in the supernatant. This explains the very high proportion of radioactivity in the supernatant when 35S was used.
The small proportion of radioactivity in the pellet can be explained by the protein coats that remain attached to the bacteria and also the presence of some flood containing protein coats in the pellet.
Landmarks in DNA research
The helical structure of DNA
If a beam of X-rays is directed at a material, most of it passed through but some is scattered by particles in the material. This scattering is called diffraction. The wavelength of X-rays makes the particularly sensitive to diffraction by the particles in biological molecules including DNA.
In a crystal the particles are arranged in a regular repeating pattern, so the diffusion occurs in a regular way. An X-ray detector is placed close to the sample to collect the scattered rays. The sample can be rotated in three different dimensions to investigate the pattern of scattering. Diffraction can be recording using X-ray film.
DNA cannot crystallise, but in 1950 Maurice Wilkins developed a method of producing arrays of DNA molecules that were orderly enough for a diffraction pattern to be obtained, rather than random scattering.
Rosalind Franklin came to work in the same research department as Wilkins. She developed a high resolution detector that produced very clear images of diffraction pattern from DNA.
From this diffraction pattern Franklin was able to make multiple deductions about the DNA structure:
- The central cross in the pattern indicated the helical shape of the molecule
- The angel of the cross shape showed the pitch (steepness of angle) of the helix
- The distance between the horizontal bars showed turns of the helix to be 3.4 nm apart
Her observations were only possible because her dedication to accuracy and perfection and were critical in the discovery of the double helix structure of DNA by Crick and Watson.
Roles of enzymes in DNA replication
- DNA gyrase moves in advance of helicase and relieves strains in the DNA molecule that are created when the double helix is uncoiled. It weakens and breaks the hydrogen bonds.
- Helicase uncoils and breaks the hydrogen bonds between nucleotides, separating the two template strands.
- Single-stranded binding proteins keep the strands apart long enough to allow the template strand to be copied.
- RNA primase puts down an RNA primer, which initiates DNA polymerase.
- DNA polymerase III adds complimentary nucleotides from 5’ to 3’ using deoxynucleoside triphosphate (dNTP).
- DNA polymerase I removes the RNA primer and replaces it with DNA. A nick is left in the sugar-phosphate backbone of the molecule where two nucleotides are still unconnected.
- Because DNA polymerase III can’t copy from 3’ to 5’, the RNA primase needs to go ahead and put down RNA primer, which RNA primase can then follow from 5’ to 3’ - Okazaki fragments.
- DNA ligase seals up the nick by making another sugar-phosphate bond.
Functions of DNA base sequences (coding and non-coding sequences)
Coding sequences (exons) are transcribed and translated when a cell requires the protein or peptide sequence that they code for.
There are also non-coding sequences, some of which have important functions:
Regulating gene expression — some base sequences are protein binding sites that either promote or repress the transcription of an adjacent gene.
Introns — They removed from mRNA before it is translated. Introns have numerous functions associated with mRNA processing.
Telomeres — these are repetitive base sequences at the ends of chromosomes. When the DNA of a eukaryote chromosome is replicated, the end of the molecule cannot be replicated, so a small section of the base sequence is lost. The presence of the telomere prevents parts of important genes at the ends of the chromosomes from being lost each time DNA is replicated.
Genes for tRNA and rRNA — transcription of these genes produces the transfer RNA used during translation and also the ribosomal RNA that forms much of the structure of the ribosome.
Bioinformatics (definition and common uses)
collecting and anaylsing complexe biological data such as genetic codes
Common uses:
locating genes that code for polypeptides within genomes, which is done using computers to search for open reading frames (ORFs).
searching for conserved sequences in the genomes of different organisms, using for finding common ancestors (less differences, more likely)
tandem repeats
Within the genomes of humans and other species, tandem repeats are regions where adjacent sections of DNA have the same base sequence.
“ACACACACACAC” (two nucleotide repeat - dimeric)
“GATGATGATGAT” (three base repeat - tetrameric)
The number of repeats varies between different individuals. These are therefore known as variable number tandem repeats. DNA profiling (fingerprinting) is based on variable number tandem repeats.
nucleosomes (+ gene regulation)
Globular structures that have a core of 8 histone proteins with DNA wrapped around.
Eukaryotic DNA – with histone proteins
Prokaryotic DNA – without histone proteins (naked DNA!) —> no nucleosomes
The eight histones in the core have N-terminal tail that extend outwards from the nucleosome. During supercoiling of chromosomes in the early stages of mitosis and meiosis the tails of histones in adjacent nucleosomes link up and pull the nucleosomes together.
During interphase, changes to the nucleosomes allow chromosomes to uncoil. The N-terminal tail are reversibly modified by adding acetyl or methyl groups. This prevents adjacent nucleosomes from packing together. The H1 histone protein (binding the DNA to the core and holding the nucleosome together) is removed so the binding of DNA to the nucleosome core is loosened. The DNA then resembles a string of beads (“Perlenkette”). Where these changes occur they allow access to the DNA by polymerase enzymes that carry out replication and transcription.
Some sections of chromosomes remain condensed during interphase and genes in these sections are therefore not transcribed. Nucleosomes thus help to regulate transcription in eukaryotes, by controlling which sections of the chromosomes are condensed or decondensed during interphase.
JMol molecular visualisation software can be used to analyse the association between protein and DNA within nucleosomes.
stages in gene expression
Gene expression is the production of mRNA by transcription of a gene and then the production polypeptides by translation of the mRNA.
promoters and transcription regulation
Gene expression can be controlled at the transcription stage.
in prokaryotes
- Control of gene expression involves a promoter, which is a base close to the start of every gene.
- The promoter itself is not transcribed, so is non-coding DNA with a function.
- RNA polymerase (RNAP) binds directly to the promoter in prokaryotes and then starts transcribing.
- Repressor proteins can bind to the promoter and prevent transcription.
in eukaryotes:
- Proteins called transcription factors bind to the promoter, which allows RNAP to bind and then initiate transcription.
- Several transcription factors are required, some of which may need to be activated by the binding of a hormone or other chemical signal.
- After transcription has been initiated, RNAP moves along the gene, assembling an RNA molecule on nucleotide at a time, from 5’ to 3’.
- Terminator sequence stops transcription at the end of the gene, separating DNA, RNA and RNAP.
identify
A polysome is a group of ribosomes moving along the same mRNA, as they simultaneously translate it.
Epigenetics (definition + evidence)
inheritance of acquired characteristics
One evidence involves small chemical marks that are attached to DNA in the nucleus of a cell to fix the pattern of gene expression. These markers are usually passed to daughter cells formed by mitosis, and help to establish tissues with common pattern of differentiation, but they are mostly erased during the gamete formation. However, a small percentage of markers persists, the epigenome, and is inherited.
Methylation and epigenetics
Cytosine in DNA can be converted to methylcytosine by the addition of a methyl group (-CH3). This change is catalysed by an enzyme and only happens where there is guanine on the 3’ side of the cytosine in the base sequence. In some eukaryotes there is widespread methylation.
Methylation inhibits transcription, so is a means of switching off expression of certain genes. The cells in a tissue can be expected to have the same pattern of methylation and this pattern can be inherited in daughter cells produced by mitosis. Environmental factors can influence the pattern of methylation and gene expression.
Fluorescent markers can be used to detect patterns of methylation in the chromosomes. Analysis of the patterns has revealed some trends:
- Patterns of methylation are established during embryo development and the percentage of C-G sites that are methylated reaches a maximum at birth in humans but then decreases during the rest of an individual’s life.
- At birth identical twins have very similar pattern of methylation, but differences accumulate during their lifetimes, presumably due to environmental differences. This is reflected in the decreasing similarity between identical twins as they age.
Post-transcriptional modification (splicing)
Eukaryotic cells modify mRNA after transcription in the nucleus. Introns are removed from mRNA before it is translated. The remaining parts of the mRNA are exons and spliced together to form mature mRNA.
Different combinations of exons can be spliced together to produce different proteins, which increases the total number of proteins an organism can produce from its genes.
Prokaryotes don’t have splicing, no exons, introns, etc. Because transcription and translation happen simultaneously.
transfer RNA (composition)
All transfer RNA molecules have:
- double-stranded sections with base pairing
- a triplet of bases called the anticodon, in a loop of seven bases, plus two other loops
- the base sequence CCA at the 3’ terminal, which forms a site for attaching an amino acid.
These features allow all tRNA molecules to bind to three sites on the ribosome — the A, P and E sites.
how transfer RNA work with amino acid attachment?
The base sequence of tRNA molecules varies, giving the molecule a destinct chemical property (unique conformation). This allows the correct amino acid to be attached to the 3’ terminal by an enzyme called a tRNA activating enzymes — one for each of the twenty different amino acids. Each of these enzymes attaches one particular amino acid to all of the tRNA molecules that have an anticodon corresponding to that amino acid. The tRNA activating enzyme recognise these tRNA molecules by their shape and chemicals properties – enzyme-substrate specificity.
ATP is needed for the attachment of amino acids to tRNA. ATP and the appropriate amino acid and tRNA bind to the active site of the activating enzyme. A pair of phosphates is released from ATP and the remaining AMP bonds to the amino acid, raising its energy level. This energy allows amino acid to bond to the tRNA. The energy from ATP later allows the amino acid to be linked to the growing polypeptide chain during translation.
