Discovery of DNA
Explain Chargaff's rules?
A% = T% and G% = C%
A+G = C+T
What is central dogma?
DNA --> RNA --> protein
Which direction is DNA read and which direction is DNA synthesized?
A 3′–OH group is exposed at the “ newest ” end of a new DNA strand; the “oldest” end has an exposed 5′ triphosphate
• DNA polymerases only assemble nucleotide chains in the 5′→3′ direction
• Because DNA strands run antiparallel to each other, the template strand is “read” in the 3′→5′ direction
explain exon shuffling
▪ It is a process through which two or more exons from different genes can be brought together ectopically, or the same exon can be duplicated, to create a new exon-intron structure. ▪ Intron-exon junctions often fall at points dividing major functional regions in encoded proteins. ▪ The functional divisions may have allowed new proteins to evolve by exon shuffling – a process by which existing protein regions or domains are mixed into novel combinations. ▪ Evolution of new proteins by this mechanism would produce changes much more quickly and efficiently than by alterations in individual amino acids at random points.
Explain the 3 types of gene regulation bacteria use
Transcriptional control—mRNA only made for proteins
needed.
▪ Translational control—Not all mRNAs are translated.
▪ Post-translational control—Proteins must be activated by
chemical modification.
Explain Griffith's Experiment and what it concluded
In 1928 Frederick Griffith found a substance that could genetically transform bacteria (transformation) – Studied two strains of Streptococcus pneumoniae:
• Smooth strain (S) is highly infective (virulent), quickly causing pneumonia and killing mice
• Rough strain (R) is nonvirulent and does not kill mice
What did Garrod's experiments conclude? Using his work what hypothesis did Beadle and Tatum develop?
Garrod- inborn error of metabolism, alkaptonuria
Beadle and Tatum- one gene-one enzyme hypothesis
What is the purpose of a sliding clamp?
• DNA polymerase extends the new DNA strand, one nucleotide at a time, by moving along the template
• Sliding DNA clamp- Protein that encircles DNA and attaches to the rear of DNA polymerase (relative to forward movement). It tethers DNA polymerase to the template strand and increases the rate of DNA synthesis
describe structure of tRNA
A transfer RNA (tRNA) brings an amino acid to the ribosome for
addition to the polypeptide chain.
▪ tRNAs are small RNAs, 75–90 nucleotides long (mRNAs are typically
hundreds of nucleotides long), with a highly distinctive structure that
accomplishes their role in translation.
▪ tRNAs wind into four double-helical segments, forming a stem-loop
cloverleaf pattern.
▪ At one end is the anticodon loop, containing the three-nucleotide
segment that base pairs with a codon in mRNAs
What is an operon?
An operon is a cluster of prokaryotic genes and associated
regulatory sequences (DNA sequences involved in the regulation
of a gene or genes).
▪ Regulatory proteins bind to these regulatory sequences to
control transcription of the genes.
▪ One regulatory DNA sequence is the promoter – the site to which
RNA polymerase binds to begin transcription.
Explain Avery's Experiment and its conclusion
In the 1940s, Oswald Avery identified the chemical nature of Griffith’s transforming principle
• Avery broke down heat-killed S bacteria and destroyed one class of molecules: Protein, DNA, or RNA
• When proteins or RNA were destroyed, the extract still transformed R bacteria into virulent S bacteria
• When DNA was destroyed, no transformation occurred – the transforming principle was DNA
What are the three steps of transcription?
initiation, elongation, termination
What are the 4 types of mutations?
Missense mutation.
▪ Nonsense mutation.
▪ Silent mutation.
▪ Frame-shift mutation
What is aminoacylation
Addition of the correct amino acid to a tRNA - aminoacylation or
charging produces an aminoacyl–tRNA.
▪ Twenty different enzymes - aminoacyl–tRNA synthetases – one
for each of the 20 amino acids – catalyze aminoacylation.
▪ The process adds free energy as the aminoacyl–tRNAs are
formed.
What proteins are produced by the lac operon? What are their functions?
β-Galactosidase.
▪ This enzyme breaks down lactose into galactose and glucose.
▪ Catalyzes the isomerization (conversion to a different form) of lactose
to allolactose, a compound that is important in regulating expression
of the lac operon.
Lactose (Galactoside) Permease.
▪ This protein, found in the E. coli cytoplasmic membrane, actively
transports lactose into the cell.
β-Galactoside transacetylase.
▪ This enzyme transfers an acetyl group from acetyl-CoA to βgalactosides.
▪ The function of this enzyme in the lac operon is not understood.
Explain Hershey and Chase's Experiment
• A phage attaches to the surface of a bacterium and infects it.
• The T2 phage studied by Hershey and Chase consists only of a core of DNA surrounded by proteins.
• They labeled either the DNA or the protein radioactively and then followed the molecule.
• They showed that labeled DNA, not labeled protein, entered the cell and appeared in progeny phages
• Bacteriophage DNA was labeled with radioactive phosphorus (32P)
• Bacteriophage protein was labeled with radioactive sulfur (35S)
• Radioactive molecules were tracked
• Only the bacteriophage DNA (as indicated by the 32P) entered the bacteria and was used to produce more bacteriophage
• Conclusion: DNA is the genetic material
describe transcription initiation in detail
Molecular machinery (RNAP & TFs) assembles at the promoter and begins synthesizing an RNA copy of the gene. ▪ The molecular machinery includes: ▪ Transcription factors (TFs) that bind to the promoter in the area of a special AT-rich sequence known as the TATA box. ▪ RNA polymerase (RNAP), an enzyme that catalyzes the assembly of RNA nucleotides into an RNA strand.
▪ The TATA box is considered a non-coding DNA sequence. ▪ It was termed the "TATA box" as it contains a consensus sequence characterized by repeating T and A base pairs.
DNA is unwound to expose the template strand – RNA polymerase II begins RNA synthesis. ▪ RNA is made in the 5′→3′ direction using the 3′→5′ DNA strand as template. ▪ When adenine appears in the DNA template strand, a uracil is paired with it in the RNA transcript.
Difference between DNA pol I and III?
DNA polymerase III is the main polymerase.
▪ DNA polymerase I is for DNA repair and helps with the lagging strand
Explain the concepts of redundancy and wobbling
Redundancy-
Given 64 possible codons and only 20 common amino acids, it is
not surprising that more than one codon specifies certain amino
acids.
Wobbling-
▪ Pairing of the anticodon with the first two nucleotides of the codon
is always precise, but the anticodon has more flexibility in pairing
with the third nucleotide of the codon.
▪ In many cases, the same tRNA anticodon can read codons that
have either U or C in the third position.
▪ The special purine inosine allows even more extensive wobble by
allowing the tRNA to pair with codons that have either U, C, or A in
the third position.
What type of operon is Trp operon? What does this mean?
The trp operon is a repressible operon – the presence of
tryptophan represses expression of tryptophan biosynthesis
genes.
▪ Tryptophan acts as a corepressor, a regulatory molecule that
activates the repressor to turn off expression of the operon (a type
of negative gene regulation).
Explain the structure of DNA (need details where everything is etc.)
Composed of nucleotides
• 5-carbon sugar called deoxyribose
• Phosphate group (PO4)- Attached to 5′ carbon of sugar
• Nitrogenous base- Adenine, thymine, cytosine, guanine
• Free hydroxyl group (—OH)- Attached at the 3′ carbon of sugar
Explain mRNA splicing and any other modifications RNA undergoes before leaving nucleus
mRNA splicing, which occurs in the nucleus, removes introns
from pre-mRNAs and joins exons together.
▪ mRNA splicing takes place in a spliceosome formed between
▪ A pre-mRNA and several small ribonucleoprotein particles
(snRNPs) – each consisting of a short small nuclear RNA (snRNA)
bound to a number of proteins.
▪ The spliceosome cleaves the pre-mRNA precisely to release the
intron and joins the flanking exons.
- further modification:
At the 5′ end of the pre-mRNA is the 5′ cap, consisting of a guanine-containing nucleotide that is reversed so that its 3′-OH group faces the beginning of the molecule. ▪ A capping enzyme adds the 5′ cap to the pre-mRNA after RNA polymerase II begins transcription. ▪ The 5′ cap (connected by three phosphate groups) is the site where ribosomes attach to mRNAs at the start of translation.
Explain the role of topoisomerase, telomerase, and telomeres.
Topoisomerase- puts nicks in DNA strands as they are being unwound to release torsional strain
Telomerase- Specialized structures found on the ends of eukaryotic chromosomes • Protect ends of chromosomes from nucleases and maintain the integrity of linear chromosomes • Gradual shortening of chromosomes with each round of cell division • Unable to replicate last section of lagging strand
Telomerase- synthesizes telomeres
What are the 5 features of the genetic code?
The code is redundant. ▪ All amino acids except methionine and tryptophan are coded by more than one codon.
▪ The code is unambiguous. ▪ A single codon never codes for more than one amino acid.
▪ The code is non-overlapping. ▪ Once the ribosome locks onto the first codon, it then reads each separate codon one after another.
▪ The code is nearly universal. ▪ With a few minor exceptions, all codons specify the same amino acids in all organisms.
▪ The code is conservative. ▪ When several codons specify the same amino acid, the first two bases in those codons are almost always identical.
Describe the impacts of Acetylation of DNA
In inactive chromatin, the histone tails are not acetylated – the tails
form a tight association with the DNA wrapped around the histone
octamer of a nucleosome.
▪ Acetylation changes the charge of the histone tails and loosens the
association of the histones with the DNA.
▪ Large multiprotein complexes bind to displace the acetylated
nucleosomes in the promoter region from the DNA or move them
along the DNA away from the promoter leading to chromatin
remodeling.