Biochemistry I - DNA Flashcards
Exceptions to the Central Dogma
- reverse transcription (RNA to DNA); reverse transcriptase is needed for the replication of retroviruses
- RNA viruses: can have their RNA translated directly to protein e.g. coronavirus, influenza virus, measles virus
- Noncoding RNA e.g. tRNA and rRNA
- Epigenetic: changes in characteristics not caused by changes in DNA sequences e.g. methylation, histone modification
DNA
*Adenine-Thymine and Guanine-Cytosine
*The human genome has 6 billion base pairs, spread over 46 chromosomes.
*phosphate group makes DNA acidic
*nitrogenous bases pair through hydrogen bonds; purines (adenine and guanine) have 2 rings and pyrimidines (cytosine and thymine) have 1
*we go from 5’ to 3’ on one side and 3’ to 5’ on the other side, making DNA antiparallel
Telomeres and single copy DNA
*telomeres are buffer zones at the ends of chromosomes that protect DNA at the ends from being lost and prevents chromosomes from sticking to each other. Telomeres normally get shorter after each replication; telomerase keeps them at the right length. If cells don’t have enough telomerase, DNA will be lost, the chromosome won’t be able to replicate and the cell will die
*Single copy DNA has most of the important genes. It is transcribed, translated and has a low mutation rate. Repetitive DNA is found near the centromeres. They may have transcribed and translated genes, but will also have genes that aren’t. Higher mutation rate. Highly repetitive DNA contains no genes, so it’s not transcribed or translated. It also has a much higher mutation rate.
* telomeres are highly repetitive DNA and genetic material is usually lost from them when Okazaki fragments are joined
DNA replication
- you can only add nucleotides on the 3’ end (from 5’ to 3’)
- topoisomerase temporarily unwinds the DNA helix and helicase breaks the hydrogen bonds between the bases on each strand
- on the leading strand: DNA primer adds a primer to the 5’ end and DNA polymerase starts to add nucleotides to the 3’ end
- on the lagging strand: DNA primer adds primers right as the helicase starts to work. DNA polymerase then adds nucleotides in the 5’ to 3’ direction (opposite direction of original helix). This forms Okazaki fragments. DNA ligase puts the strand together and the RNA primers are replaced with DNA.
- DNA polymerase works as fast as 700+ base pairs per second FAST
- there is 1 mistake for every 10 million nucleotide; proofreading occurs, reducing this to 1 mistake for every billion nucleotides PRECISE
- out of conservative, dispersive and semiconservative models, Meselson and Stahl proved DNA replication is semi conservative.
Transcription (DNA to mRNA) - in nucleus
- RNA polymerase binds to a promoter. Every gene has a promoter. The RNA polymerase then separates the strand forming single stranded templates and then adds nucleotides complementary to the template strand it’s focusing on. It does this from the 5’ to the 3’ end.
- the RNA being coded will be complementary to the template strand and so similar to the coding strand with the only difference being uracil replacing thymine.
- this process ends when the RNA polymerase reaches a terminator. The mRNA that’s coded might form a hairpin which impairs the RNA polymerase’s function.
- in eukaryotes, the pre-mRNA is going to be processed by adding a 5’ cap (modified guanine) and a poly-A tail at the 3’ end. This protects the ends of the mRNA. The pre-mRNA is also spliced, removing introns (don’t encode the genes we’re focusing on) and leaving only the exons.
Translation (mRNA to protein) - in cytoplasm
- a ribosome is made up of proteins (60S and 40S) and ribosomal RNA.
- AUG is a START codon; 61 out of the 64 possible codons code for amino acids and 3 codons are STOP codons.
- each tRNA can bind to specific amino acids on one end and have anticodons on the other end that pair with the appropriate codon.
- the appropriate tRNA initially binds at the A (amino acyl) site. Another tRNA will then bind on the A site while the first tRNA shifts to the P site. A peptide bond will form between the two amino acids they carry,the ribosome will shift to the right placing the first tRNA in the E site (exit site) and the second in the P (polypeptide chain forms here) site. The polypeptide stays with the tRNA moving to the P site while the first tRNA molecule in the E site is ejected. This continues until you get to the STOP codon where the polypeptide is released.
- in prokaryotes: non coding region, shine Delgado sequence (ribosomal binding site), non coding region and then START codon. Translation occurs until STOP codon, then non coding region
- in eukaryotes: 5’ cap (ribosomal binding site), non coding region, START codon. Translation occurs until STOP codon, then non coding region, then poly-A tail 100-250 nucleotides). The 5’ cap and poly-A tail prevent mRNA from being degraded by enzymes. Prokaryotes don’t have this because both transcription and translation both happen in the same place and can happen at the same time. mRNA in eukaryotes travel and so need the extra protection
- in prokaryotes, the first amino acid is formyl-methionine. In eukaryotes, it’s a methionine.
DNA repair
Exonuclease activity (during replication):
-DNA polymerase III replicates and proofreads. It can sense if it makes mistakes, goes back (3’to 5’), removes the incorrect base and replaces it with the correct one. It can only remove this from the end of the strand which is why it’s, an exonuclease. DNA polymerase I is an exonuclease in the 5’ to 3’ direction.
Mismatch repair mechanism (after replication):
- proteins recognise a mismatch due to distortion in sugar backbone. They mark the area with a cut. An exonuclease will then remove the incorrect nucleotide, and one of the DNA polymerase inserts the right nucleotide. DNA ligase connects the new nucleotide with nucleotides on the same strand and complementary nucleotide. The proteins know the parent strand in bacteria because it is methylated.
DNA damage (structure of DNA):
-UV rays (cause the formation pyrimidine dimers - protrusion in sugar backbone), Gamma rays, X-rays
- reactive oxygen species (superoxide anion, peroxides)
- nucleotide excision repair gets rid of pyrimidine dimers: endonuclease cuts out dimer and any other incorrect ones. DNA polymerase brings the right nucleotides and DNA ligase creates all necessary bonds
*a very damaged cell might go into senescence, apoptosis or unregulated cell division
Protein modification
- Co-translational e.g. acetylation (first amino acid is removed and replaced with an acetyl group)
- Post-translational modification: more common; occurs in the ER and Golgi
- glycosylation: adding carbohydrates to a protein; e.g. in ABO groups
- lipidation: adding a lipid to a protein
- phosphorylation: adding a phosphate group to a protein or enzyme by a kinase e.g. phosphorylation of sodium potassium pumps to change their conformation and release contents
- methylation e.g. methylation of histones that turns genes on and off
- proteolysis: you might need to cut a protein to activate it e.g. zymogen
- ligands: adding a ligand to give protein function e.g. Ubiquitination
Jacob Monod lac Operon
Gene expression is normally regulated at the level of transcription because this is most efficient. We’re not making any RNA or protein we don’t need
Lac operon is found in bacteria and it has codons that code for enzymes that help bacteria break down lactose; lac z (beta galactosidase) lac y (lactose permease), lac A.
- these genes are not expressed when lactose isn’t present. After the promoter site and before the start site, there is an operator site with a repressor. It blocks RNA polymerase from transcribing and uses glucose as an energy source
- when there’s lactose, it binds to the repressor and makes it come off the operator site. RNA polymerase will transcribe all the genes needed to metabolise lactose
- when the lactose level goes down and even the lactose on the repressor is metabolised, the conformation of the repressor changes again and it rebinds to the operator.
- it is the presence of lactose that induced the transcription of the operon.
- if glucose and lactose are present, glucose binds to lac Y and prevents it from bringing in any more lactose. Allolactase cause a conformation change in the repressor, allowing for transcription
