Molecular Genetics Flashcards
Purines
Double Ringed
A, G
Pyrimidines
Single Ring
C, T
DNA Structure
Turns clockwise, complete turns every 10 nucleotides
5’ ends with open phosphate
3’ ends with open OH
C numbered clockwise (1= right of oxygen)
-N base bonds to carbon one with glycosyl bond
-C 5 and C 3 bond with phosphodiester bond
Virus Structure
- Single molecule of DNA/ RNA surrounded by protein coat
- Can be single or double stranded DNA or single stranded RNA
- DNA forms loops
DNA in Prokaryotes
Circular DNA packed in one area of cell (nucleoid)
-Loop of DNA twisted into supercoiled structure
Have small circular DNA called plasmids- carry genes such as antibiotic resistance
Levels of Chromosome packaging
1) DNA wrapped around histones (proteins)-> nucleosome
2) Nucleosomes fold back and form chromatin
3) DNA compresses when dividing, chromatin forms folded loops that attach to protein scaffold
4) Protein scaffold folds into chromosomes
Conservative Model
DNA stays intact, new double strand made using old strand as template
Results in 1 molecule of new DNA, one old
Semi Conservative Model
DNA splits into strands, each strand paired with matching nucleotides
Results in 2 strands with half old, half new
Meselson and Stahl proved this in 1957 using N isotopes to track DNA replication
Dispersive Model
DNA breaks into small fragments and reassembles with mixture of old and new on both sides
DNA Replication- Initiation
DNA Helicase unwinds helix by breaking H bonds starting at the origin
SSB (Single Stranded Binding Proteins) keep strands apart
DNA Gyrase releases tension from unwinding by cutting and reforming bonds
DNA Replication- Elongation
2 Helicase working in opposite direction, creates 2 replication forks
Primase places RNA primer of a few nucleotides
DNA polymerase III attaches new nucleotides (can only work in 5-3 direction- leading strand, built towards replication fork)
Other side replicated in discontinuous fashion (lagging strand)
Lagging Strand
-Built away from replication fork
RNA primers placed by primase
Okasaki fragments of DNA built in 5-3 direction from one RNA primer to the other using polymerase III
-DNA polymerase I codes over RNA primer and replaces it with DNA (leaves a gap in backbone), also proofreads
DNA ligase joins okizaki fragments by joining phosphodietster bonds
DNA Replication- Termination
-2 molecules made
Primers removed by DNA polymerase I
-Ligase fills in gap b/w Okazaki fragments
-Exonuclease (enzyme) cuts out wrong nucleotides and adds correct ones when mistakes occur
Central Dogma of Protein Synthesis
- DNA = plan
- Copy of DNA (RNA)= action
Protein Synthesis- Transcription- Initiation
1) Initiation- RNA polymerase binds to DNA and opens double helix
- RNA binds upstream” of gene at region called promoter (TATA box).
Protein Synthesis- Transcription- Elongation
2) Elongation- RNA polymerase builds mRNA in 5’-3’ direction
- No primer required
- template strand= transcribed side
- coding/nonsense strand- side not copied
- mRNA complimentary to template, identical to coding
Protein Synthesis- Transcription- Termination
- RNA polymerase recognizes end when it comes across terminator sequence and releases template strand of DNA
- RNA breaks away
- RNA polymerase now free to bind to another gene
- Several RNA polyermase can be transcribing a gene at the same time
Protein Synthesis- Transcription- mRNA modification
1) Capping and Tailing
- 5’ cap added (7 methyl guanosine- modified guanine nucleotide triphosphate)
- Poly-A tail added (200-300 adenine ribonucleotides added to 3’ end by poly A polymerase enzyme
2) mRNA splicing
- introns (non coding regions) removed by spliceosomes
Protein Synthesis- Translation- Initiation
- mRNA binds to rRNA on small ribosomal subunit
- tRNA with anticodon UAC binds to mRNA/rRNA romplex (carried methionine)
- aminoacyl-tRNA synthetase charges tRNA by adding a.a.
- Binds to large subunit
Protein Synthesis- Translation-Elongation
a.a. binds to A site
large subunit creates peptide bond with previous a.a.
-Ribosome moves along mRNA
-Used tRNA released at E site which frees A site
Protein Synthesis- Translation-Termination
Protein release factor binds to stop codon in site A
Cleaves polypeptide from tRNA, breaks apart ribosome subunits
Polysome
1 mRNA bound to more than one ribosome at the same time
Post Translational Modification
- Some a.a. removed
- Polypeptide divided into pieces
- sugar, phosphate added
- forms quaternary structure with other polypeptides
Transcriptional control
Proteins determine when/how often RNA polymerase can bind
Post Transcriptional control
Modifications to pre-mRNA affect life/use of RNA in cytoplasm
Post Translational control
Proteins activated through processing
Signals enlist cell machinery to destroy proteins
Control Region (promoter)
Responds to presence/absence of lactose and glucose
Coding Region
Produces enzymes needed to digest lactose
Activator
fast/slow switch
Operator
on/off switch
Repressor Protein
binds to operator and prevents RNA polyermase from binding
When lactose is present it binds to represser, changes 3D shape and repressor removed from operator
Cyclic AMP
Detects glucose levels
Catabolite Activator Protein (CAP)
cAMP binds to CAp, together they bind to DNA ahead of promoter to help RNAP bind
cAMP activated CAP proteins stimulate transcription of 100+genes including lac operon
RNAP can still work without cAMP-CAP complex but is less likely to bind therefore slower
Point Mutations
Silent-Doesn’t change a.a. b/c codes for same a.m
Mis-sense- Incorrect a.a, can cause malfunctioning protein
Non-sense- Codes for stop a.a, shortens protein
Frameshift Mutations
Insertion- nucleotide inserted
Deletion- nucleotide deleted
Chromosomal Mutations
Deletion- removes chromosomal segment
Duplication- repeats a segment
Inversion- reverses a segment within chromosome
Translocation-one segment from one chromosome moved to another
Telomere Control
Telomere- molecular cap (repeating TTAGG) that shortens with every division b/c cells cant replicate it easily
Once too short, gene p53 stops cell replication
Telomerase rebuilds telomere, allows cell to keep dividing
Friedrich Miescher
Investigated composition of DNA using pus cells
Discovered nuclei of cells contained large quantities of substance unlike proteins (named nuclein)
Hammerling
Discovered that cells with nucleus removed could not regenerate but cells with nucleus but other part removed could
Griffith
Injected mice with encapsulated and uncapsulated pneumonia cells
epacsulated= death
uncapsulated= alive
Avery, McCarty, McLeod
Ruptured heat killed encapsulated cells to release contents tested RNA, DNA, protein coats for transforming activity
Result- DNA is transforming principle, not proteins
Hershey, Chase
Virus has 2 parts (DNA, Protein coat), only DNA enters bacteria when infecting, not protein coat
Restriction Endoculease
enzyme that is able to cleave double stranded DNA into fragments at specific sequences
Sticky ends
end of DNA fragment w/o complimentary base (has overhang)
-Easier to connect
Blunt ends
DNA fragment with complimentary base (no overhang)
PCR
-Polymerase Chain Reaction
-direct method of making copies of a desired DNA sequence easily (doesn’t have to be inserted into a plasmid)
-
PCR process
- Heat breaks DNA into 2 strandes
- Temperature lowered
- DNA primers replace RNA primers
- Taq polymerase adds complimentary nucleotides
- Process repeats
Gel Electrophoresis
- Separates DNA fragments by size
- solution containing DNA fragments is placed in a well in the gel, which is a rectangular or square slab that contains electrolytes. A negative charge is placed at one end of the gel (where the wells are) and a positive charge is placed at the other end of the gel. The negatively charged DNA moves towards to positive charge. The smaller fragments move faster than the larger fragments, creating separation
Gene cloning
- A gene can be cloned into a plasmid when a gene fragment that was cut by the same restriction enzyme that cut a plasmid joins with the plasmid fragment. DNA ligase reforms the phosphodiester bond between the fragments, and the plasmid as circular again and contains the foreign gene fragment. When the plasmid replicates, each copy will contain both the plasmid fragment and the foreign gene fragment, therefore, the gene has been cloned.
