Chapter 7: Molecular Genetics Flashcards
DNA
- ATCG
- hereditary information of the cell
- double helix w/major and minor grooves
- backbone 5’ to 3’ phosphodiester bond form phosphate backbone
- CUT the PYE-C,T, and U are pyrimidines, A and G are purines
RNA
- AUGC
- function usage varies per type
- mRNA liner=provides the instructions for assembling amino acids into a polypeptide chain
- tRNA clover= delivers amino acids to a ribosome for their addition into a growing polypeptide chain
- rRNA globular=combines with proteins to form ribosomes
DNA replication
- second chromatid containing a copy of DNA is assembled during interphase
- DNA is unzipped and each strand serves as a template for complementary replication
- semiconservative replication= one strand is old and the other is new
helicase
- enzyme that unwinds DNA, forming a Y shaped replication fork
single stranded binding proteins
- attach to each strand of uncoiled DNA to keep them separate
topoisomerases
- break and rejoin the double helix, allowing the prevention of knots (if you unwind a twist, the ends will get extra tight and knot up)…. ahead of the replication forlk
DNA polymerase
- move from the 3’-5’ direction ONLY, synthesizes a new strand that is antiparallel (5’-3’)
leading strand
works continuously as more DNA unzips (synthesized 5’ to 3’)
lagging strand
for the 5’ to 3’ template strand the DNA polymerase has to go back to the replication fork and work away from it. It produces fragments at a time called okazaki fragments vs continuous replication
DNA ligase
connects okazaki fragments
primase
an enzyme that creates a small strip of RNA primer off of which DNA polymerase can work since it can only add to an existing strand
- ** DNA replication required an RNA primer
- **every okazaki fragment has an RNA primer which are later replaced with DNA by DNA Pol 1
DNA Pol 1
- replaces base pairs from the primer and does DNA repair
- has 3-5’ exonuclease: breaks phosphodiester backbone on a single strand of DNA and removes nucleotide
- also has 5’ to 3’ exonuclease, to take primer off, and can also proof with 3’ to 5’ when laying down new chain
DNA Pol 3
- is pure replication
- has 3-5’ exonuclease: breaks phosphodiester backbone on a single strand of DNA and removes nucleotide
- proofreading… if it makes mistake it will go back and use this to replace it
Random facts
- in prokaryotes … the “good “ strand is methylated after replication so it doesn’t accidentally repair wrong strand
- in all cases of repair, DNA ligase must come in to seal the backbone afterward
energy for elongation
- provided by two additional phosphates attached to each new nucleotide (making a total of three phosphates attached to the nitrogen base)
- breaking the bonds holding the two extra phosphates provides chemical energy for the process (same w/transcription! Human rate is 50n/s
replication of telomere
two problems occur
- not enough template strand where primase can attach
- Last primase is removed=> in order to change RNA to DNA, there must be another DNA strand in front of the RNA primer> DNA pol cant build after removing RNA primer > ultimately that RNA is destroyed by enzymes that degrade RNA left on the DNA, section of the telomerase subsequently lost w/each replication cycle.
* ** Prokaryotic DNA is circular so no telomere (or issue)
telomerase
- enzyme that attaches to the end of the template strand and extends the template by adding short sequence of DNA over and over (not important code), allowing elongation to continue
- However, at the end will still be not enough for primase to attach but this loss of unimportant segment will not cause any problem
- telomerase carries an RNA template: binds to flanking 3’ end of telomere that compliments part of its RNA template, Synthezies to fill in over the rest of its template
protein synthesis
- **note: one-gene-one-polypeptide hypothesis defines a gene as the DNA segment that codes fora particular peptide
2. genetic code is universal for nearly all organisms and most amino acids have more than one codon specifying them (redundancies/degeneracies)
mRNA
- single stranded template. Since there are 64 possible ways (4x4x4) that four nucs can be arranged in triplet combos, there are 64 possible codons. 3 are stop codons… therefore 61 codes for a.a.
tRNA
C-C-A-3’ end of the t-RNA attaches to amino acid, and other portion is the anticodon which bp with the codon in mRNA
wobbles:
exact bp of 3rd nuc in the anticodon and the 3rd nuc in the codon is often not required, allowing 45 different tRNA’s to base-pair with 61 codons that code for amino acid.
2. Transports AA to its mRNA codon
rRNA
- nucleolus is an assemblage of DNA actively being transcribed into rRNA
- as ribsomes, has three binding sites: one for mRNA, one for tRNA that carries a growing polypeptide chain (p Site); one for 2nd tRNA that delivers the next aa (A site)
- Termination sequences include UAA, UGA, UAG
- together w/ proteins, rRNA forms ribosomes. Ribosome is assembled in nucleolus but large and small subunits exported separately to cytoplasm
Transcription
- creation of RNA molecules from DNA template… prokaryotes are polycistronic (more than one polypeptide per RNA molecule) and eukaryotes are monocistroninc (encode only one polypeptide per RNA molecule)
Transcription: initiation
- RNA pol attaches to promoter region on DNA and unzips the DNA into two strands
- a promoter region for mRNA transcription often contains the sequence TATA (TATA Box)
- Most common sequence of nucs at promoter region is called the consensus sequence; variation from it cause less tight RNA pol binding— lower transcription rate
Transcription: elongation
- RNA pol unzips DNA and assembles RNA nucleotides using one strand of DNA as template; only one strand is transcribed
Transcription: termination
- when RNA pol reaches a special sequence often AAAAA in eukaryotes
* **note: transcription is occurring in the 3’ to 5’ direction of the DNA (but sunthesis of RNA strand is 5 to 3)
mRNA processing
before leaving the nucleus, pre-mRNA undergoes several modifications
5’ cap (-P-P-P-G-5’)
- the sequence is added to the 5’ end of the mRNA
2. guanine with 2 phosphate groups=>GDP; providing stability for mRNA and point of attachment for ribosmes
poly-A tail (-A-A-A-A…..A-A-3’)
- sequence is attached to the 3’ end of mRNA
- Tail consists of 200A; provide stability and control movement of mRNA across the nuclear envelope (in prokaryotes… poly A tail facilitates degradation!)
RNA splicing
- removes nucleotide segments from mRNA
- before mRNA moves into cytoplasm, small nuclear ribonucleoproteins (snRNP’s) and the spliceosome delete the introns and splice the exons (prokaryotes have no introns!)
alternative splicing
- allows different mRNA to be generated from the same RNA transcript; by selectively removing differences of an RNA transcript into different combinations => each coding for a different protein product
* ** note: in prokaryotes generally have ready-to-go mRNA upon transcription .. it is only in eukaryotes that you need the above processing; because prokaryotes do not need to process their mRNA first, translation can begin immediately/simultaneously
* *** in both prokaryotes eukaryotes, multiple RNA polymerases can transcribe the same template simultaneously.
translation
- assembly of amino acids based on reading of new mRNA; uses GTP as energy source
aminoacyl-tRNA
in cytoplasm, amino acid attaches to tRNA at 3’ end, require 1 ATP —> AMP per AA
translation: initiation
- small ribosome unit attaches to 5’ end of mRNA
- tRNA-methionine attaches to start sequence of mRNA AUG
- large ribosomal unit attaches to form a complete complex (requires 1 GTP)
translation: elongation
- next tRNA binds to A site, peptide bond formation, tRNA without methionine is released
- the tRNA currently in A site moves to P site (TRANSLOCATION) and the next tRNA comes into A site and repeat process (requires 2 GTP per link)
translation: termination
- encounters stop codon UAG, UAA, UGA
- polypeptide and two ribosomal subunits all release once release factor breaks down the bond between tRNA and final AA of polypeptide
- while polypeptide is being translated, AA sequences is determining folding conformation; folding process assisted by chaperone proteins (requires 1 GTP)
post-translation
- translation begins on a free floating ribosome
- signal peptide at the beginning of the translated polypeptide may direct the ribosome to attach to the ER, in which case the polypeptide is injected into the ER lumen
- if injected, polypeptide may be secreted form the cell via Golgi
- In general, post-translational modifications (addition of sugars, lipids, phosphates groups to the AAs may occur)
- may be subsequently processed by the golgi before it is functional
clarifications about translation
- amino acids are placed starting for the 5’ end of the mRNA and move all the way down to the 3’ end
- tRNA anticodons are 3’ to 5’
- can occur simultaneously with transcription in prokaryotes but not eukaryotes
- multiple ribosomes may simultaneously translate 1 mRNA
* *** in bacteria, the start codon is n-formylmethionine rather than methionine
mutation
- if error is not repaired, it becomes a mutation
- sequence of nucleotides in a DNA molecule that does not exactly match the original DNA molecule from which it was copied
point mutation
is a single nucleotide error and includes the following
- substitution
- deletion
- insertion
- frameshift occurs as a result of a nucleotide deletion or insertion
silent mutation
new codon still codes for the same amino acid
missense mutation
new codon codes for NEW amino acid=> minor or fatal results as in sickle cell
nonsense mutation
new codon codes for a stop codon
neutral mutation
no change in protein function
repair mechanisms
- proofreading: DNA polymerase checks base pairs
- mismatch repair: enzymes repair things DNA polymerase missed
- excision repair: enzymes remove nucleotides damaged by mutagens
DNA organization
- nucleosome= DNA is coiled around bundles of 8/9 histones (beads on a string)
- Not during division, chromatin exists as either of two types: Euchromatin, heterochromatin
euchromatin
loosely bound to nucleosomes, actively being transcribed
heterochromatin
areas of tightly packed nucleosomes where DNA is inactive (condensed=> darker) … contains a lot of satellite DNA (larger tandem repeats of noncoding DNA)
transposons (jumping genes)
- DNA segments that can move to new location one same/different chromosome… 2 types
- insertion sequences consist of only one gene that codes for enzymes that just transports it (transposase)
- complex transposons code for extra: replication, antibiotic resistance, etc,
- Insertion of transposons into another region could cause mutation (little to no effect)
virus
a nucleic acid (RNA/DNA may be double or single stranded)
- capsid= protein coat that encloses the nucleic acid
- capsomeres=assembles to form the capsid
- envelope= surrounds capsid of some viruses… it incorporates phospholipid/protein obtained from cell membrane of host
* ** bacteriophage =virus that only attacks bacteria
* *** usually specific to a type of cell (bind to specific receptors) and species
* ** host range is range of organisms virus can attack
replication: lytic cycle
- virus penetrates cell membrane of host and uses host machinery to produce nucleic acids and viral proteins that are assembled to make new viruses- these viruses burst out of the cell and infect other cells
DNA virus
DNA is replicated and form new viral DNA => transcribed to produce viral proteins (DNA + viral proteins assemble to form new viruses)
RNA virus
RNA serves as mRNA=> translated into protein (protein + RNA=> new virus)
retroviruses
HIV, ssRNA (single stranded) viruses that use reverse transcriptase to make DNA complement of their RNA => which can go to manufacture mRNA or go into lysogenic cycle (becoming incorporated into DNA host)
lysogenic cycle
- viral DNA is incorporated into DNA of host cell
- dormant state (provirus/prophage [if bacterial]); remain inactive until external stimuli .. when triggered beings lytic cycle
prions
not viruses of cells, but misfolded versions of proteins in the brain that cause normal version to misfold too. Fatal
molecular genetics of bacteria
- bacteria are prokaryotes with no nucleus or organelles
- single circular ds DNA molecule (tightly condensed and called a nucleiod)
- no histones or other associated proteins
- replicate DNA in both directions from single point of origin (theta replic.)
- reproduce by binary fission= chromosomes replicates, cell divides into two cells, each cell bearing one chromosome)
- lack nucleus=> lack mictrotubules, spindle, centrioles
plasmids
- short, circular DNA outside chromosome
- carry genes that are beneficial but not essential for survival)
- replicate independently
episomes
are plasmids that can incorporate into bacterial chromosome
conjugation
donor produces a bridge (pilus) and connect to recipient; send chromosome/plasmid to recipient and recombinant can occur, F plasmid allowing pilus to occur; once recipient receives, it is now F+ and can donate as well. R plasmids provide bacteria with antibiotic resistance
— pili also used for cell adhesion
transduction
DNA is introduced into genome by a virus
2. when virus is assembled during lytic cycle, some bacterial DNA is incorporated in place of viral DNA. Virus infects another host, the bacterial DNA part that it delivers can recombine with the resident DNA
transformation
bacteria absorb DNA from surroundings and incorporate into genome
regulation of prokaryotic gene expression
operon=control gene transcription, consist of …
- promoter: sequence of DNA where RNA polymerase attaches to begin transcription
- operator: region that can block action of RNA poll if occupied by repressor protein
- structural genes: DNA sequences that code for related enzymes
- regulatory genes: located outside of operon region, produces repressor proteins, other produce activator proteins
Lac operon (E.coli)
- controls breakdown of lactose
- regulatory gene produces active repressor (bound to operator) and blocks RNA pol
- when lactose is available, lactose binds to repressor and inactivates it=> RNA pol can now transcrive
- lactose induces the operon
- the enzymes that the operon produces are said to be inducible enzymes
**Note: consists of three lac genes (Z, Y, A) which code for B-galactosidase (convert lactose into glucose/galactose, lactose permease (transport
actose into cell), and thiogalactoside transacetylase. Also know that low glucose means high cAMP levels cAMP binds to CAP binding site
of promoter RNA polymerase more efficiently transcribes lactose can be broken down. If lactose AND glucose are high, operon is shut
off (cAMP is low, doesn’t bind to CAP, bacteria uses one sugar at a time and prefers glucose).
trp operon (e.coli)
- produces enzyme for tryptophan synthesis
- regulatory genes produce inactive repressor=> RNA pol produces enzymes
- when tryptophan is available, no longer need to synthesize it internally: it binds to inactive repressor and activates repressor=> able binds operator and block RNA pol.
- tryptopham is corepressor
respressible enzymes
as above, when structural genes stop producing enzymes only in presence of active respressor
— unlike repressible enzymes, some genes are constitutive (constantly expressed) either naturally or due to mutation
regulation of eukaryotic gene expression
- regulatory proteins: repressors and activators, influence RNA pol’s attachment to promoter region
- nucleosome packing: methylation of histones (tighter packing = prevent transcription); acetylation of histones (uncoiling and transcription proceeds)
- RNA interference: short interfering RNAs (siRNAs)= block mRNA transcriptions (fold back within itself=dsRNA), translation, or degrade existing mRNA:
- –siRNAs: dsRNA gets chopped up then made single stranded. the relevant strand will bind to DNA ( prevent transcription) or mRNA (signals destruction)
human genome
- 97% of human DNA does not code for protein product; noncoding DNA: regulatory sequences, introns, repetitive sequences never transcribed, etc. Tandem repeats abnormally long stretches of back to back repetitive sequences within an affected gene (e.g. Huntingtons)
recombinant DNA
- contains DNA segments or genes form different sources
- the transfer of these DNA segments can come from viral transduction, bacterial conjugation, transpososns, or through artificial recombinant DNA technology
- crossing over during prophase of meiosis produces recombinant chromosomes
recombinant DNA technology
- uses restriction endonucleases to cut up specific segments of DNA and left it with sticky end (unpaired)
- these restriction enzymes (eg. EcoRI; BamHI) normally used by bacteria to protect against viral DNA (protect their own DNA methylation)
vector
such as plasmid because DNA molecule used as vehicle to transfer foreign genetic material into another cell
- to introduce foreign DNA into plasmid, the plasmid is treated with the same restriction enzyme so the same sticky ends bind
- – DNA ligase stabilizes the attachments; then the plasmid is introduced into bacterium by transformation
- –after this process, bacterial can grow to produce product, form clone library, etc
- – use antibiotic resistance/screen method to filter out the ones that don’t have the recombinant DNA
gel electrophoresis (after DNA cut up)
- agarose gel under an electric field for the separation of proteins based on charge and size
- –negative DNA moves towards positive anode from negative cathode
- –shorter DNA moves further than larger; distributes DNA by size
restriction fragment length polymorphism (RFLPs)
restriction fragments between individuals are compared, fragments differ in length are observed because of polymorphism (different length in DNA sequences)
—- inherited in Mendelian fashion so often used in paternity suits and crime scenes to match suspects
DNA-finger printing
RFLPs at crime scene compared to RFLPs of suspects
short tandem repeat (STR)
repeat of 2-5 nucleotides and different between all individuals except identical twins
reverse transcriptase
introns often prevent transcriptions; this enzyme makes DNA molecule directly from mRNA. DNA obtained from this manner is complementary DNA (cDNA) which lacks introns that suppress transcriptions
polymerase chain reaction (PCR)
uses synthetic primer (the primer may be RNA or DNA oligonucleotides) to clone DNA (rapidly amplify).
