Bio 3 Flashcards
In what direction is DNA synthesized?
5’ to 3’
What does helices do? What does it use?
helicase unwinds DNA, it uses ATP to do this
What does topoisomerase do?
decreases the tension in DNA as helices unwinds it
What do single-stranded binding proteins do?
help stabilize and keep DNA separated once it has been unwound
What is the primosome? What does it do?
it includes primase, an RNA polymerase that makes primers for DNA replication
What is the replisome?
it includes DNA polymerase, is used in DNA replication
What is ligase?
connects DNA fragments
What causes the Hayflick Limit?
the shortening of telomeres
How much do telomeres shorten by each replication?
50-200bp
What are the 2 options for telomeres during replication? What cells normally do what?
replicate telomeres using telomerase - usually germ cells, embryonic stem cells and many cancerous cells
or
stabilize telomeres, ~300nt of ssDNA at the chromosome ends form loops and bind proteins
What is telomerase? How does it work?
it is a ribonucleoprotein that uses an RNA template and reverse transcriptase to synthesize telomeres in 6nt repeats
Do bacteria have telomeres?
no because their chromosomes are circular
What is theta replication?
prokaryotic DNA replication because they just have one ori per chromosome and their chromosomes are circular
How is ori recognized in prokaryotes?
DnaA
How is ori recognized in eukaryotes?
3 proteins that make up the origin replication complex (ORC)
How many components are in the prokaryotic replisome? Eukaryotic?
prokaryotic = 13 eukaryotic = 27
How many DNA polymerases do prokaryotes have? Eukaryotes?
prokaryotes= 5- I, II, III, IV and V eukaryotes= many, we don't need to know them
What is the main DNA polymerase used in prokaryotic replication? What are some of its features?
DNA polymerase III
it has high processivity and 3’ to 5’ exonuclease activity (proofreading)
What is DNA polymerase II used for?
back up to DNA polymerase III
has 3’ to 5’ exonuclease acivity
What is DNA polymerase I used for? What are some of its features?
has poor processivity
it has 5’ to 3’ exonuclease activity
it removes RNA primers and puts in the right DNA
also used in repair
What are DNA polymerases IV and V used for?
mostly for repair and stalling other polymerases at replication forks
they are error prone
What is heterogeneous nuclear RNA?
hnRNA is a precursor to mRNA in eukaryotes
it is hnRNA before it is processed i.e. cap, tail and splicing
What is snRNA?
small nuclear RNA is part of the spliceosome
What are miRNA and siRNA?
micro RNA and small interfering RNA
they function to control gene expression regulation
What is piRNA?
PIWI-interacting RNAs are single-stranded and short
work with PIWI proteins to prevent transposons from mobilizing
What is long ncRNA?
longer than 200 nts
help control gene expression and contribute to many types of post-transcripitonal regulation
In which direction does transcription occur?
5’ to 3’
Does RNA polymerase require a primer for transcription?
no
In which direction is the template strand read for transcription?
3’ to 5’ (because its synthesized 5’ to 3’)
What are the sense and antisense strands?
antisense is the template strand
sense is the coding strand
How many types of RNA polymerase do prokaryotes have?
they have one type of RNA polymerase that makes all types of RNA
Describe prokaryotic RNA polymerase
core enzyme (the part that does elongation) has 5 subunits- 2 alpha, beta, beta', omega holoenzyme (needed to initiate transcription) also contains another subunit called the sigma factor
Describe prokaryotic promoters
have 2 primary sequences
the Pribnow box at -35
and the -35 sequence
What unwinds double-stranded DNA in transcription?
RNA polymerase does it itself
What is the closed complex in transcription? What is the open complex?
closed complex is when the DNA hasn’t been unwound yet
open complex is when it has been unwound and can now be transcribed
Describe the sigma factor’s role in prokaryotic transcription?
increases the ability of RNA polymerase to recognize promoters and decreases non-specific affinity of the holoenzyme for DNA
after transcription has started it is no longer needed and dissociates from the RNA polymerase complex
How are the location of eukaryotic and prokaryotic transcription different?
prokaryotic transcription occurs in the cytosol and is thus simultaneous with translation
eukaryotic transcription occurs in the nucleus
What is eukaryotic RNA polymerase I used for?
most rRNA
What is eukaryotic RNA polymerase II used for?
hnRNA (ultimately mRNA), most snRNA and some miRNA
What is eukaryotic RNA polymerase II used for?
tRNA, long ncRNA, siena, some miRNA and a small subset of rRNA
How does splicing work?
a splice some that contains small ribonucleic particles (snRNPs) made from proteins and snRNA assemble around each intron
they recognize and h-bond to 5 conserved residues:
GU at the 5’ end
AG at the 3’ end
and an A 15-45 bases upstream from the 3’ splice site
Can genes have more than one promoter?
yes they can for alternative splicing
In what 3 ways is eukaryotic hnRNA modified to become mRNA?
splicing
5’ cap
3’ poly-A-tail
What is the 5’ cap on mRNA? What is it essential for?
it is a methylated guanine
necessary for translation
also important in prevent mRNA digestion by free exonucleases in the cell
What is the poly-A-tail? What is it needed for?
several hundred As added to the end of eukaryotic mRNA
important in preventing mRNA digestion by free exonucleases in the cell
What is an IRES?
internal ribosome entry site
found in some eukaryotic mRNA transcripts
allows for cap-independent translation
Describe eukaryotic promoters
have core promoters at -50
TATA box at -25
upstream control elements at -200
What is the amino acid acceptor site on tRNA?
always CCA at the 3’ end of the tRNA molecule
What is Wobble Hypothesis?
the 3rd position of a tRNA anticodon is flexible (the first 2 need to be correct though)
How many ATP equivalents are needed for tRNA loading/amino acid activation?
2 ATP equivalents
put AMP onto AA and then remove the AMP while adding tRNA
What are aminoacyl-synthetases?
enzymes that recognize the amino acid and tRNA that should be together
they are very specific and have a low error rate
Describe the prokaryotic ribosome
70S ribosome
has 50S and 30S subunits
large subunit (50S) has 23S and 5S RNA molecules and 31 peptides
small subunit (30S) has 16S RNA and 21 peptides
23S is the main ribozyme
Describe the eukaryotic ribosome
80S ribosome 60S and 40S subunits 60S subunit has 5S 5.8S and 28S RNA with 46 peptides 40S has 18S RNA and 33 peptides 28S is the main ribozyme
Where is ribozymic activity found in the ribosome?
in the large subunit for both eukaryotes and prokaryotes
What are the 3 sites in a ribosome?
EPA
E= exit site
P= peptide site
A= aminoacyl site
In what direction does translation occur?
5’ to 3’
What is the ribosome binding site in prokaryotes? Where is it found?
Shine-Delgarno sequence at -10 upstream of the start codon in the 5’UTR
it is complementary to a pyrimidine-rich region in the small subunit
Where do humans inherit mtDNA from?
from their mother only, because sperm only contribute nuclear DNA to a zygote
What is the transcription stop signal?
in prokaryotes there is a transcription stop sequence
in eukaryotes its the poly-A tail
Describe prokaryotic initiation (of translation)
small ribosomal subunit (30S) binds IF1 and IF3
this then binds the mRNA
next the f-Met tRNA joins along with IF2 which is bound to GTP
this initiator tRNA sits in the P site of the ribosome (70S) and is H-bonded to the start codon
the 50S subunit then joins and is powered by the hydrolysis of GTP
before elongation begins all of the initiation factors dissociate
What is f-Met?
formylmethionine
the first amino acids used in all prokaryotic proteins
In which direction is a protein synthesized?
N terminus to C terminus
Describe prokaryotic elongation (in translation)
an aminoacyl-tRNA enters the A site which requires the hydrolysis of GTP by EF-Tu
EF-Ts then removes the GDP from EF-Tu
the peptidyl transferase activity of the large ribosomal subunit (due to 23S rRNA) then catalyzes the the formation of a new peptide bond
EF-G (also called translocase) helps with translocation of the empty tRNA into the E site and the growing peptide into the P site, using a GTP in the process
EF-Tu eventually helps remove the tRNA from the E site
and the cycle can repeat
Describe prokaryotic termination (of translation)
when a stop codon appears the the A site a release factor binds to it
RF1 recognizes UAA and UAG
RF2 recognizes UAA and UGA
RF3 hydrolyzes GTP and leads to dissociation of RF1 and RF2 after peptide release
ribosome separates and mRNA and peptide are released
What stop codons does RF1 recognize?
UAA and UAG
What stop codons does RF2 recognize?
UAA and UGA
How many high-energy bonds are required to make a peptide?
4n with n being the number of amino acids in the chain
What is the translation initiation sequence in eukaryotes?
Kozak sequence
located a couple nucleotides before the start codon in the 5’UTR
Describe initiation of translation in eukaryotes
43S pre-initiation complex forms: 40S subunit, Met-tRNA and several eIFs
Complex is then recruited to the 5’ capped end of the transcript by an initiation complex of proteins
It then scans from the 5’ end looking for a start codon
Once the start codon has been found the 60S subunit binds
Describe elongation of translation in eukaryotes
eEF-1 helps with the entry of an aminoacyl-tRNA and has a subunit that helps it release GDP after GTP hydrolysis
eEF-2 is the eukaryotic translocase
Describe termination of translation in eukaryotes
eRF1 recognizes all 3 stop codons
eRF3 is a ribosome-dependent GTPase that helps eRF1 release the peptide
Does DNA methylation turn on or off eukaryotic gene expression?
turns it off
What is genomic imprinting?
when only one allele of a gene is expressed
involves DNA methylation, histone modification and binding of long ncRNAs
How does X-chromosome inactivation occur in human females?
at the blastocyst stage all of the cells in the inner cell mass randomly inactivate one X chromosome
every cell derived from each cell will have the same X inactivated, but because they are done randomly different tissues etc may have different ones
Is an enzyme for an anabolic pathway more likely to be repressible or inducible? What is the default setting for this type of system?
repressible
default is “on”
Is an enzyme for a catabolic pathway more likely to be repressible or inducible? What is the default setting for this type of pathway?
inducible
default is “off”
What is an example of a repressible operon?
trp operon
codes for an enzyme for tryptophan anabolism
What is an example of an inducible operon?
lac operon
codes for an enzyme for lactose catabolism
What are the 3 genes in the lac operon?
Z, Y and A
What does gene Z encode in the lac operon?
the enzyme beta-galactosidase, which cleaves lactose in glucose and galactose
What does gene Y encode in the lac operon?
permease, a protein which transports lactose into the cell
What does gene A encode in the lac operon?
transacetylase, an enzyme which transfers acetyl groups from acetyl-CoA to beta-galactosides (not required for lactose metabolism)
What two genes are not actually in the lac operon, but are important for its regulation? What do they encode?
crp gene - codes for the catabolite activator protein (CAP) and helps couple the lac operon to glucose levels in the cell
I gene - codes for the lac repressor protein
Describe how glucose levels regulate the lac operon
when glucose is low, adenylyl cyclase is activated and cAMP levels are high
cAMP binds to CAP and this complex binds to the promoter of the lac operon helping activate RNA polymerase
Describe how the lac repressor protein regulates the lac operon
repressor binds to the operate if there is no lactose present and blocks transcription
lactose allosterically binds to the repressor protein if it is present and this stop the repressor from binding to the operator, allowing for transcription
What will bacterial cells preferentially use as an energy source?
glucose
Describe how the trp operon works
repressor protein is encoded by the trpR gene
when tryptophan is present it binds to the repressor and together they bind the operator, blocking transcription
when tryptophan is not present repressor protein can’t bind an transcription can occur
What kind of viruses can have envelopes?
animal viruses
For the MCAT are viruses considered living?
no
What is another name for attachment and penetration in the viral reproductive cycle?
adsorption and penetration
How do viruses that infect bacteria get their genome inside the cell?
attach via receptor binding and insert the genome only
the capsid stays outside
What is the capsid of a virus?
its protein outershell
How do viruses that infect animal cells get their genomes inside the cell?
through receptor-mediated endocytosis
or through receptor-mediated fusion (if they have an envelope)
the viral genome is then uncoated once inside
Explain the virus lytic cycle that involves hydrolase expression
virus expresses early genes and takes over host
virus expresses viral hydrolase which fragments the host genome (into dNTPS)
What kind of viruses can express viral hydrolase? Do they all express it?
only lytic viruses can express viral hydrolase, but they don’t all express it
Explain the viral lytic cycle that does not involve hydrolase
virus expresses early genes and takes over host cell
virus then hijacks host metabolic pathways, but host genome stays intact
virus then expresses late genes (viral genome replication, transcription, translation and viral assembly)
virus then kills host cell, uses lysozyme commonly in bacterial cells, however this won’t work for animal cells because they don’t have a cell wall (use other methods)
What kind of viruses can undergo the lytic cycle?
bacterial and animal
What kind of viruses can undergo the productive cycle?
animal only
Explain the viral productive cycle
virus expresses early genes and takes over host cell
then hijacks host metabolic pathways
expresses late genes
host cell doesn’t die, viruses “bud” out of host cell- this is how animal viruses can get an envelope
What kind of viruses can undergo the lysogenic cycle?
bacterial or animal
Explain the lysogenic viral cycle
viral genome is integrated into host genome
viral genome is silent because of viral-encoded repressor
cell undergoes cell cycle, viral genome is replicated along with host genome and provirus/prophage is activated
viral genome excision occurs (can take some host DNA with it = transduction)
virus then proceeds to either the lytic or productive cycle
What is the genome of a host cell that has had a lysogenic virus integrate its genome called when it is still silent?
provirus (animal)
prophage (bacterial)
What is transduction?
when a lysogenic virus takes some of the hosts DNA with it when it leaves and gives it to another cell
What are the 6 types of viral genomes?
\+ssRNA -ssRNA retrovirus (+RNA that do lysogenic) dsRNA ssDNA dsDNA
What is a positive RNA strand?
has the same polarity as mRNA
Which types of viruses have an “infective genome”?
all of them do except for -ssRNA (need to carry an RNA-dependent RNA polymerase with them)
Describe what a +ssRNA virus does and what is required for it
encodes an RNA-dependent RNA polymerase which replicates the genome
host can make the required proteins
Describe a -ssRNA virus does and what it requires
encodes an RNA-dependent RNA polymerase, but also needs to carry one with it
RDRP transcribes the genome and it can them be translated
Describe a retrovirus and what it requires
+ RNA that do the lysogenic cycle
encode an RNA-dependent DNA polymerase (reverse transcriptase)
RdDP is translated and then it can convert the genome into dsDNA
dsDNA then integrates into host genome
Describe a dsRNA virus and what it requires
encodes an RNA-dependent RNA polymerase which replicates its genome
host can make the required proteins
(same as +ssRNA)
Describe a ssDNA virus and what it requires
doesn’t require anything :) some will encode genes to promote repair, nucleotide synthesis, cell cycle etc
host cell “repairs” ssDNA to dsDNA it is then transcribed and translated
Describe a dsDNA virus and what it requires
doesn’t require anything :) some encode genes to promote repair, cell cycle etc
limiting factor is the cell cycle of host cell
virus can push host through cell cycle or carry replication machinery
How are prions and viroids exceptions to the central dogma?
prions are self-replicating proteins
viroids are self-replciating RNA molecules
What is a prion?
infectious misfolded proteins
resistant to heat and degradation
hard to destroy
What is a viroid?
infectious circular +ssRNA molecules
200-400nts
have high self-complementarity
What is the function of a viroid?
are non-protein-coding
can be ribozymes?**
siRNA
What can prions cause?
BSE (mad cow disease)
CJD
are transmitted by ingesting nervous tissue from infected host
How do viroids replicate?
RNA-dependent RNA polymerase produces -RNA which is used to make copies of the +RNA
are circularized via RNA ligase
can hijack host cell machinery (mechanism isn’t known)
What can viroids cause?
many plant diseases
HepD in humans
What is required for viroids to infect a human with HepD?
needs to connect with HepB for capsid and enzymes
