Molecular Biology Flashcards
Why are DNA and RNA called nucleic acids?
they are found in the nucleus and possess many acidic phosphate groups
What is the building block of DNA?
deoxyribonucleoside 5’ triphosphate (dNTP)
What are the three components of deoxyribonucleotides?
- ribose (simple monosaccharide)
- aromatic base (A, G, C, T)
- phosphate group
What modification makes ribose special?
the 2’ OH is missing, so it is deoxyribose
Why are the aromatic molecules A, G, C, T bases?
they contain several nitrogens which have free electron pairs capable of accepting protons
Purines? Pyrimidines?
GA (two rings)
CUT
What is a nucleoside?
ribose with a purine or pyrimidine linked to the 1’ carbon in a Beta-N-glycosidic linkage
In the Beta-N-glycosidic linkage of a nucleoside, is the aromatic base above or below the plane of ribose in a Haworth projection?
above
Will A and T H-bond with each other in a dilute aqueous solution?
no they will be H-bonded with water
What is the key determinant of the double stranded structure of DNA?
H-bonds
Do DNA bases interact with water?
no because the DNA coiling places them inside the double helix where they interact with each other
Nucleotides are _________ of nucleosides.
phosphate esters
Where does the phosphate group attach on the ribose ring?
5’ OH group
What is called when nucleotides contain three phosphates?
deoxynucleoside triphosphates (dNTP)
What is the backbone? Why?
ribose + phosphate because it doesnt change
What is the backbone in protein, and what is the variable portion?
peptide bonds with a carbon between them
R group
What is a deoxynucleoside?
just the ribose and the aromatic base
Nucleotides in DNA chain are covalently linked by _______.
phosphodiester bonds between the 3’ hydroxy groups of one deoxyribose and the 5’ phoshphate group of the next deoxyribose
Which reaction is more thermodynamically favorable: the polymerization of nucleoside monophosphates, or the polymerization of nucleoside triphosphates?
triphosphate because pyrophosphate is released and hydrolyzed driving the reaction forward
What is a polymer of several nucleotides linked together?
oligonucleotide or poly
A polynucleotide is named in what direction?
5’ to 3’
In AGCT, which has the the free 5’ and 3’ groups?
A
T
What common protein structure often depends on H-bonds between anitparallel chains?
beta pleeted sheets in secondary structure
how many nucleotides long is a kbp?
1,000
What is annealing, or hybridization?
the binding of two complementary strands of DNA into a double stranded structure
what is melting, or denaturation?
separation of strands
What is the Tm?
the temp at which a solution of DNA molecules is 50% melted
If you attached methyl groups to all the acidic phosphate oxygens along the length of a DNA double helix, would the chain have a higher or lower Tm than normal DNA?
the methyl groups are non charged meaning they will not repel each other so it will take more kinetic energy to melt them so Tm would be higher
The two hydrogen bonded antiparallel DNA strands form a ______ handed double helix with the bases on the ______ and the ribose/phosphate backbone on the _______.
right
interior
exterior
What stabilizes the double helix?
- Van der Waals forces between the bases, which are stacked on each other
- hydrophobic interactions
How far away from each other are the bases when they are stacked?
3.4 angstroms
The helix pattern repeats itself once every ___ angstroms, which is every ___ base pairs.
34
10
What is the width of the double helix?
20 angstroms
What is one angstrom in meters?
10^-10 meters
What is the genome?
sum total of an organism’s genetic info
Each piece of double stranded DNA is called a ______.
chromosome
Prokaryotic genomes are composed of a __________.
single circular chromosome
T/F
Viral genomes are always circular.
False
they can be linear or circular
The human genome consists of ___ base pairs while the bacterial genomes consist of ____ base pairs.
10^9
10^6
What is the cause of the size difference between human genomes and bacterial genomes?
it is the result of repetitive DNA that has no know function, not due to evolutionary sophistication
What is the role of DNA Gyrase?
it uses the energy of ATP to twist the gigantic circular molecule in prokaryotes to make DNA more compact and sturdy
How does DNA Gyrase function?
by breaking the DNA and twisting the two sides of the circle around each other (supercoils)
What is Eukaryotic DNA wrapped around to package it tighter?
histones
DNA with the appearance of beads on a string?
nucleosomes
Nucleosomes are composed of DNA that are wrapped around an _____ of histones.
octamer (8 histones)
The string between the beads is a length of double helical DNA called ______ and is bound by a single _______.
linker DNA
linker histone
Fully packed DNA is called _____.
chromatin
Are histones basic or acidic?
basic (Arg,Lys) because they are attracted to the acidic exterior of DNA double helix
If you start with a deoxyribose what do you add to get a nucleoside?
base
What do you add to nucleoside to get a nucleotide?
three phosphates
How do you get an oligonucleotide from a nucleotide?
polymerize with loss of two phosphates
How do you go from a single stranded polynucleotide to double stranded DNA?
two complete chains H-bond in antiparallel orientation
How do you go from a ds-DNA to a ds-helix?
coiling
How do you go from a ds-helix to a nucleosome?
wrap around histones
What is the next step in DNA packaging after nucleosomes?
complete packaging into chromatin
Process of converting DNA to RNA?
Transcription
Process of converting RNA to protein?
Translation
Central Dogma?
DNA to RNA to Protein
Summary of process of converting DNA to protein?
- DNA is copied to mRNA
- mRNA travels to cytoplasm where it encounters ribosomes which contain many proteins (rRNA)
- ribosome synthesizes polypeptides to make protein according to DNA’s original orders
What are the three stop codons?
UAA
UAG
UGA
How many codons are there? How many specify amino acids?
64
61
3 stop
What is another name for stop codons?
nonsense codons
What is the term for two or more codons coding for the same amino acid?
synonyms
T/F
Each codon specifies only a single amino acid
True
Intercalating?
inserting themselves between base pairs
Mutagen?
any compound that can cause mutations
What are the three kinds of mutations?
- point
- insertion
- deletion
What is a point mutation?
single base pair substitution
What are the two types of point mutations?
- transitions- ex. sub of a purine for another purine
2. transversions- sub a purine for pyrimidine
What are the three subclasses of point mutations?
- missense- one amino acid is replaced by different amino acid
- nonsense- stop codon replaces a regular codon
- silent- codon is changed to another codon for the same amino acid
If a missense mutation leads to little change in the structure and function of the gene product, it is called a _______.
conservative mutation
What can insertions and deletions cause?
frameshift mutations- shift in the reading frame
Are all insertions and deletions frameshift mutations?
no not if a whole codon is replaced by another codon
Replication occurs during the ______ in interphase of the cell cycle.
S phase
Semiconservative?
1 parental, 1 daughter together
Conservative?
2 parental together
2 daughter together
Dispersive?
mixture of parent and daughter strands
T/F
DNA replication is semiconservative.
True
What does DNA polymerase do?
catalyzes the elongation of the daughter strand using the parental template
What is the thermodynamic driving force of polymerizatioin?
the removal and hydrolysis of pyrophosphate from each dNTP added to the chain
Polymerization (replication) occurs in what direction?
5’ to 3’
The template strand is read in what direction?
3’ to 5’
T/F
DNA pol does not require a template.
False
T/F
DNA pol requires a primer.
True
Where does the primer for DNA pol come from?
an RNA polymerase called Primase begins DNA replication by creating a small RNA primer that DNA pol can elongate by adding deoxyribonucleotides to the existing primer, the primer is later replaced by DNA
What enzyme unwinds the DNA and separates the strands to start replication?
Helicase
Would helicase use ATP to separate the strands?
yes because separating the strands requires breaking many H-bonds
How is the origin of replication found?
enzymes scan for the right nucleotide sequence
What enzyme relives the stress of coiling on either side of the replication fork brought on by helicase?
Topoisomerase
What protein keeps the strands separated?
single stranded binding proteins (SSBP)
After the strands have been separated, what is the first step to start replication?
synthesis of RNA primer on each template strand
How does DNA pol elongate primer?
by adding dNTPs to its 3’ end
T/F
Replication proceeds along in both directions away from the origin.
True
The leading strand is synthesized ______ while the lagging strand is synthesized ______.
continuously
discontinuous
What chunks of DNA comprise the lagging strand?
Okazaki fragments
After RNA primers are replaced by DNA, the fragments are joined by what enzyme?
DNA ligase
What is the difference in the origin of replication between prokaryotes and eukaryotes?
Eukaryotes have many origins or replication bubbles because their chromosomes are huge whereas Prokaryotes only have one origin
Prokaryotic replication is also know as ________.
Theta replication
How many DNA polymerases are in prokaryotes?
3
What is the function of DNA polymerase III?
responsible for synthesizing the leading strand very fast in the 5’ to 3’ direction
Can DNA pol III correct its mistakes? how?
yes it can chop off the nucleotide it just put on in a process known as proofreading using a 3’ to 5’ exonuclease activity
Difference between exonuclease and endonuclease?
Exo- cuts at the end
Endo- cuts in the middle
What are two important types of endonucleases?
- Repair enzymes- remove chemically damaged DNA from the chain
- Restriction enzymes- to destroy DNA of infecting viruses
What is the role of DNA pol II?
unknown
What is the role of DNA pol I?
remove the primer and replace it with DNA
DNA pol I has _____ exonuclease activity.
5’ to 3’
What are three ways that RNA is chemically distinct from DNA?
- single stranded
- contains uracil instead of thymine
- pentose ring is ribose instead of deoxyribose
What makes RNA less stable than DNA?
2’ OH group in RNA is highly reactive, DNA has a 2’ H
Why does RNA have uracil and DNA has thymine?
thymine is easier for DNA repair systems to work with, RNA is less energy costly which makes it easier for RNA to move around
Why are drugs that block thymine used for cancer therapy?
thymine is only used in replication, so if it is blocked only replication is inhibited and the rest of the cell can function
mRNA?
tRNA?
rRNA?
mRNA- carries genetic info from the nucleus to the cytoplasm for translation
tRNA- translates genetic code
rRNA- components of the ribosome, catalytic function of ribosome
Eukaryotic mRNA is ____cistronic.
mono
Monocistronic?
one gene, one protein
there are as many different mRNAs as there are proteins
Prokaryotic mRNA is ___cistronic.
poly
How many different tRNAs are there?
20, one for each amino acid
T/F
Both replication and transcription involve template driven polymerization.
true
Transcription occurs in the ______ direction.
5’ to 3’
Does RNA pol require a primer for transcription?
no
Does RNA pol have exonuclease ability?
no it cannot correct its errors
Transcription is a _____ fidelity process than replication.
lower
what is the name of the place where transcription starts?
the start site
What activates RNA pol to begin transcription?
promoter
T/F
Transcription is the principle site of the regulation of gene expression in just eukaryotes meaning that the amount of each protein made in every cell is controlled by the amount of RNA that gets transcribed.
False
in both eukaryotes and prokaryotes
What are some names for the strand that is transcribed?
template, non coding, transcribed, antisense
it is complementary to the transcript
What are some names for the DNA strand that is not transcribed?
coding, sense
it is the same sequence as the transcript but with T instead of U
Transcription proceeds upstream or downstream?
downstream
Downstream?
Upstream?
- toward 3’ end of coding strand and transcript (+)
- toward 5’ end of coding strand, beyond transcript (-)
In prokaryotes, all types of RNA are made by the _____ RNA polymerase.
same
How many subunits does prokaryotic RNA polymerase have? whats it called?
5
2 alpha, 1 beta, 1 beta’, 1 omega
called the core enzyme
What is the core enzyme responsible for in prokaryotic transcription?
rapid elongation of the transcript
What is the role of the sigma factor in prokaryote transcription?
required to form the holoenzyme which is responsible for initiation
What are the three stages of transcription?
- Initiation
- Elongation
- Termination
Initiation occurs in prokaryotic transcription when ______.
RNA pol holoenzyme binds to a promoter
What are the typical promoters in prokaryotic transcription?
- 10 box (Pribnow)
- 35 sequence
Holoenzyme scans along the chromosome until it recognizes a promoter and then stops, forming a _______.
closed complex
When can transcription begin in prok?
once the open complex has formed by RNA pol binding to the promoter with a region of single stranded DNA
What two roles does the sigma factor play in helping the polymerase find promoters?
- greatly increase the ability of RNA pol to recognize promoters
- decrease the nonspecific affinity of holoenzyme for DNA
The core enzyme elongates the RNA _______, with one pol complex synthesizing an entire RNA molecule.
processively
As the core enzyme elongates the RNA, it moves along the DNA ______ in a transcription bubble.
downstream
What happens when a termination signal is detected (Rho) in prok transcription?
pol falls off of DNA, releases the RNA, bubble closes
T/F
tRNA and rRNA are polypeptides.
False
gene products
The transcription of anabolic pathways should be ______ by their product.
inhibited
The transcription of catabolic pathways should be inhibited whenever ________.
their substrate is not around, and activated when it is.
Anabolic enzymes are________. Catabolic enzymes are ______.
repressible
inducible
What are the 4 main differences between prok and Euk transcription?
- location of transcription
- primary transcript and mRNA
- RNA pol
- regulation
Karyon?
nucleus
Location of transcription Prok vs Euk?
Prok- occurs in cytoplasm, transcription and translation can occur simultaneously
Euk- occurs in nucleus, then RNA is modified, transport across nuclear membrane to cytoplasm where translation can occur, does not occur simultaneously
Difference in RNA pol Prok vs Euk?
Prok- RNA pol complex (aBB’sigmaomega)
Euk- three different RNA pols
What are the roles of the three RNA pol in Eukaryotic transcription?
- transcribe rRNA
- transcribe mRNA
- transcribe tRNA
T/F
One difference is in Euk the transcript is ready to be translated immediately, whereas the Prok transcript needs to be modified.
False
its the other way around
What is the most important example of the Euk transcript is modified before translation?
splicing
Euk mRNA has intervening sequences that do not code for anything that are called ______.
introns
The regions of Euk mRNA that actually get expressed are called ______.
exons
What is splicing?
the process of removing introns and joining exons together
What type of RNA is found in the nucleus that is the primary transcript made by RNA pol II, before splicing?
heterogeneous nuclear RNA (hnRNA)
What is the 5’ cap? 3’ poly A tail? importance?
- 5- cap- methylated guanine nucleotide stuck on the 5’ end, important for translation, preventing digestion of the mRNA by exonucleases that are free in the cell
- Tail- string of several hundred adenine nucleotides, important in preventing digestion of the mRNA by exonucleases that are free in the cell
Why would exonucleases be floating free in the cell?
- mRNA has a very short lifespan
2. virus injected it
How can there be synthesis of two different polypeptides from one piece of RNA?
splicing (hnRNA)
In prok transcription, what is the problem with having some promoters stronger than others as a mechanism of regulation?
it is pre set so it does not respond to change
What advantage is there in turning off the genes when the protein products arent required if they are not harmful?
it requires a lot of energy to synthesize RNA and protein, so it is more efficient to transcribe what is needed
What type of enzymes are used in anabolic processes? why?
repressible because transcription is inhibited in the presence of excess amounts so they dont keep building more up
What type of enzymes are used in catabolic processes? why?
inducible because transcription is stimulated when there is an abundance of substrate
What two components does the Lac operon have?
- coding sequence for enzymes
2. upstream regulatory sequences (control sites)
What two regulatory sequences does the Lac operon contain?
the promoter and the lac operator
What is the purpose of transcription in the Lac operon?
DNA is transcribed to RNA which is translated to make three enzymes used for lactose catabolism
What is the role of the repressor in Lac operon?
sits on the operator and prevents RNA pol from binding to the promoter and transcribing
What happens when lactose is high in the cell in Lac operon?
lactose is converted to allolactose which binds to the repressor causing a conformational change, the repressor falls off the operator, RNA pol can bind and transcription proceeds
What happens when lactose is low in Lac operon?
there isnt enough to bind to the repressor so transcription is stopped
If the operator is mutated so that the lac repressor can no longer bind, what effect will this have on transcription?
nothing will stop RNA pol from transcribing so it will continue
If a mutation in a Euk fat cell reduces the level of several proteins related to fat metabolism, does this mean the proteins are encoded by the same mRNA?
no, Euk are monocistronic meaning one mRNA can encode for only one protein
Basic summary of translation?
mRNA attaches to a ribosome at a specific codon, the specific amino acid is delivered by a tRNA, then the second amino acid is delivered by another tRNA, the ribosome binds them together to get a dipeptide, it continues until the polypeptide is complete
What is the anticodon?
sequence of three ribonucleotides that is complementary to the codon the tRNA translates
T/F
base pairing between the tRNA anticodon and the mRNA codon are specific.
True
Do the three nucleotides in the anticodon contribute to the tertiary structure of the tRNA?
no the anticodon needs to be ready to base pair with the codon
Where is the amino acid attached to the tRNA?
amino acid acceptor site
If you looked at many tRNA molecules, which region would you expect to vary the most?
the anticodon because it is different on every tRNA molecule so it can bind to its specific amino acid
Describe the thermodynamics and kinetics of peptide bond formation during protein synthesis. What is used to power this process?
it is unfavorable (G>0) and slow (high Ea)
reaction coupling
If the amino acid acceptor sites are the same, how is the attachment of the appropriate amino acid accomplished?
aminoacyl-tRNA synthetase enzyme
How does aminoacyl-tRNA synthetase enzyme recognize the correct amino acid?
it recognizes the tertiary structure of both the amino acid and the tRNA
What is it called when the amino acid is attached to the tRNA?
amino acid activation
What is used for the activation of each amino acid?
two high energy phosphate bonds (equals 2 ATP)
Prok Ribosome? Two subunits in cytoplasm?
70S
50S and 30S
Euk Ribosome? Two subunits in cytoplasm?
80S
60S and 40S
What are the three binding sites in the complete ribosome (both subunits together)?
- A site- where each new tRNA delivers its amino acid
- P site- where the growing polypeptide chain, still attached to a tRNA, is located during translation
- E site- where the now empty tRNA sits prior to its release from the ribosome
In Prok, does translation always begin at the 5’ end?
since it is polycistronic, there is more than one translation start site so they cant all be at the end
What is the Shine Dalgarno sequence in Prok?
ribosome binding site which acts as a promoter for initiation of translation (-10 upstream)
Initiation of translation in Prok requires the formation of _____.
70S initiation complex
What makes up the 70S initiation complex in Prok?
30S subunit
mRNA
first aminoacyl tRNA
50S subunit
What powers the process of the formation of the 70S complex?
hydrolysis of one GTP
What is the initiator tRNA in Prok?
fMET-tRNA or mylmethionine which is a modified amino acid used as the first amino acid in all Prok proteins
What is the start codon?
AUG
What are the three steps in elongation in Prok translation?
- the 2nd aminoacyl tRNA enters the A site and H-bonds with the 2nd codon (takes 1 GTP)
- peptidyl transferase catalyzes the formation of a peptide bond between fMET and the second amino acid
- translocation- tRNA 1 moves into the E site, tRNA 2 moves into the P site, next codon moves into the A site (takes 1 GTP)
What happens if tRNA binding to the E site is disrupted?
increase in the number of frameshift mutations in resulting protein
When does termination occur in Prok translation?
when a stop codon appears in the A site, then a release factor enters the A site
How many high energy phosphate bonds are required to make a 50 amino acid polypeptide chain?
199
costs about 4n or 50 X 4= about 200
What is the direction of translation in prok?
N to C
What is different about Euk translation as opposed to Prok?
- ribosome is larger
- translation and transcription cannot occur simultaneously
- N terminal amino acid is different (Met instead of fMet)
- splicing, capping, tailing
- do not use shine dalgarno sequence to initiate, uses Kozak sequence
What processes can occur simultaneously in Euk?
transcription and splicing because they both occur in the nucleus
A competitive inhibitor of Euk RNA pol III would have the greatest effect on _______. Why?
translation
because RNA pol III transcribes tRNA
T/F
In Euk mRNA, transcription stops at the stop codon.
False
translation stops at the stop codon, transcription stops when it is signalled
Which is less accurate, transcription or replication?
transcription
What do telomeres do?
- lengthen the ends of DNA
- protect the ends of the chromosomes from damage due to incomplete replication
What would a longer half life of RNA lead to?
more protein would be translated because it would stay in the cell longer
