Unit 2 - Molecular Genetics (2) Flashcards
Conservative model
both strands of the original DNA molecule remain intact and together
a new copy of the DNA molecule is made with new nucleotides
Semi-conservative model
two original DNA strands separate and are used as a template for the synthesis of new complementary strands from new nucleotides
one strand of the new DNA molecules is made of old DNA
Dispersive model
the DNA molecule is fragmented and the old fragments are put back together with new fragments made from new nucleotides
all strands of the new DNA molecules have some of the old DNA
Meselson and Stahl
DNA molecules can be separated by their mass using centrifugation in a test tube of cesium chloride solution
DNA containing N-14 doesn’t get pushed as far as DNA containing N-15
Meselson and Stahl Experiment #1
they grew bacteria on agar medium containing only N-15 for several generations, so that the bacteria contained only heavy DNA
these bacteria were transferred to agar containing only N-14 and allowed to grow for one generation (DNA will be copied once)
the DNA after one cell division was extracted and centrifuged
result = single band of medium DNA was observed
conclusion = conservative method does not occur, since a heavy and light band were not observed
Meselson and Stahl Experiment #2
some of the bacteria from experiment 1 were allowed to grow on N-14 for two generations
the DNA of these bacteria was extracted and centrifuged
results = two bands of DNA, one medium and one light in density
conclusion = disproves the dispersive model, as the DNA molecules would have had to been the same density
DNA replication
occurs during interphase
DNA polymerase enzymes (different variations) replicate DNA and also proofread the sequence as they go
DNA replication starts at an origin of replication, which is rich in A and T pairings, making the strands easier to separate
circular bacterial chromosomes and plasmids only have one origin of replication
eukaryotic chromosomes are much longer and have multiple origins spaced about 300 000 base-pairs apart
Steps of DNA replication
DNA polymerase uses the RNA primers made by primase as a starting block
the primers are lengthened using deoxynucleoside triphosphates (dATP, dGTP, dCTP, dTTP) from the cytoplasm that are complementary to the next DNA nucleotide on the complementary strand
the nucleotide insertion removes the two extra phosphates, powering the DNA synthesis
DNA polymerase reads the strand from 3’ to 5’, but writes a complementary strand from 5’ to 3’
since the two DNA strands are antiparallel, this means that DNA replication differs on the two strands of a replication fork
leading strand is formed in one piece
lagging strand forms from smaller pieces called Okazaki fragments
the Okazaki fragments are subsequently glued together by ligase
when DNA polymerase reaches an RNA primer, it uses its proof-reading function to replace the primer with DNA nucleotides
Additional enzymes for DNA replication
helicase = unwinds the double helix at an origin by breaking hydrogen bonds, forming a replication fork
gyrase/topoisomerase = as the double helix is unwound, it becomes supercoiled in front of the replication fork, so gyrase cuts one strand to eliminate the problem
single strand binding proteins (not enzymes) = prevents the separated DNA strands from recombining prematurely
Beadle and Tatum experiment
studied bread mold
the wild-type strain grows on minimal medium because it can synthesize the amino acid arginine using a metabolic pathway
three different mutants were identified which were each unable to synthesize arginine, each had a defect at a different reaction in the pathway
each mutant was able to grow on minimal medium if it was enriched by the product of its defective reaction
they concluded that each mutation was an abnormal gene, and that each gene directs the production of an enzyme
this experiment led to the one gene-one enzyme hypothesis
it is now known that genes code for polypeptides, only some of which become enzymes
The genetic code
only 4 bases are used to specify 20 amino acids
the genetic code must have units of at least 3 bases
amino acids in a polypeptide are specified by base triplets called codons
the message only makes sense using the correct order and reading frame of codons
the genetic code is essentially universal, only a few obscure codon exceptions have been observed
Transcription
RNA polymerase recognizes the TATA box of a promoter, just in front of the gene
proteins called transcription factors are sometimes required to help RNA polymerase bind to the promoter
RNA polymerase separates the DNA strands at an initiation site in the promoter
RNA polymerase moves away from the promoter, reading the template strand of DNA from 3’ to 5’ and synthesizes a complementary messenger RNA out of RNA nucleotides from 5’ to 3’
the DNA strands reform a double helix after RNA polymerase has passed
at a termination site, at the end of the gene, RNA polymerase is released, along with a primary transcript of the gene
Background for RNA processing
heating DNA breaks the hydrogen bonds holding the strands together so they separate
cooling the separated strands allows them to recombine
cooling the separated strands along with mRNA allows the mRNA to base-par with the gene from which it is derived, forming a hybrid molecule
RNA splicing
the average length of transcribed DNA is 8000 nucleotides
the average polypeptide is only 400 amino acids long
long non-coding DNA segments (introns) are found between expressed regions (exons) in genes
the initial RNA transcript is oversized and never leaves the nucleus
spliceosomes made of RNA and protein snip out the introns and splice the eons to form a finished mRNA that can enter the cytoplasm for translation
intron splicing may help control gene expression
splicing introns in different ways could allow the same gene to code for different proteins (contrary to one gene-one polypeptide hypothesis)
RNA processing in eurkaryotes
RNA molecules are modified before leaving the nucleus
the 5’ end is capped with a modified GTP
this may prevent attack by ribonucleases, assist the binding of small ribosomal subunits, and facilitate exit from the nucleus
about 200 adenine molecules are added to the 3’ end
poly A tail is a sacrificial extension that is broken down by ribonucleases to give the mRNA time to make protein at ribosome
