What is the central dogma of molecular biology?
DNA makes RNA, RNA makes protein
What are the two kinds of nucleic acid?
DNA and RNA
How do DNA and RNA differ?
in chemical composition and structure
- RNA is single-stranded; DNA double
- RNA is made of ribose; DNA of deoxyribose (missing an OH group that RNA has)
- RNA contains uracil; DNA contains thymine
Who first suggested a double-helical model for the structure of DNA?
James Watson and Francis Crick
How did they figure out that DNA must have a double-helix structure?
best way molecule could fit together from the evidence they had (including x-ray crytallography)
What was the chemical evidence about the structure of DNA?
puring and pyrimiding nigrogenous bases; deoxyribose sugar, phosphate
What is the biological evidence about the structure of DNA?
ratios between bases (proportion of A = T; C = G)
What is the physical evidence about the structure of DNA?
x-ray crystallography; 2 nm diameter, double helical structure
What does the fact that nitrogenous bases are located on the inside of a DNA molecule indicate about their nature?
they’re relatively hydrophobic
What deductions did evidence from Rosalind Frankland’s work enable Watsom to make?
- DNA was helical
- width of the helix
- spacing of the nitrogenous bases
- nitrogenous bases on inside
What is Chargaff’s rule?
the proportion of A = T; C = G
Does A + C have to equal G + C?
no
What are the two types of nitrogenous basees?
pyrimidines and purines
What is the difference between a pyrimidine and a puring?
purines are made of two carbon-nitrogen ring bases (4 N in total); pyrimidines are made of one nitrogen ring base (2 N in total)
What type of nitrogenous base is adenine?
purine
What type of nitrogenous base is thymine?
pyrimidine
What type of nitrogenous base is guanine?
purine
What type of nitrogenous base is cytosine?
pyrimidine
What two components of a DNA molecule interact to form the phosphate-sugar backbone of DNA?
the free 3’-OH group of deoxyribose in the 1st nucleotide and the 1st 5’ phosphate in the 2nd nucleotide (5’P - 3’OH)
How many nucleotides may be present in a single DNA molecule?
many millions
How many strands is a DNA molecule made of?
2
How are the two strands of a DNA molecule oriented with respect to each other?
antiparallel (one strand goes from 3’ to 5’; other from 5’ to 3’)
What are the base pairings in a DNA molecule?
A:T and C:G
How many hydrogen bonds are present between adenine and thymine?
2
How many hydrogen bonds are present between cytosine and guanine?
3
In what direction does a DNA molecule grow?
from 5’ to 3’ (must add 5’P to free 3’ OH)
What do restriction enzymes do?
break DNA phosphodiester bond at a recognized “binding site”
How did evidence indicate that a purine and a pyrimidine had to pair (instead of purine-purine or pyrimiding-pyrimidine)?
process of elimination - knew that the pairing had to be 2 nm wide; only pair that fit that was purine-pyrimidine?
How did evidence indicate that A paired with T and C paired with G?
only pairings that allowed bases to hydrogen bind
What would the diameter of a DNA molecule be if a pyrimidine bonded to another pyrimidine, and how does that relate to the X-ray data?
less than 2.0 nm (too thin compared to X-ray data)
What would be the diameter of a DNA molecule if purines bonded to pyrimidines, and how does that relate to the X-ray data?
larger than 2.0 nm (too thick compared to X-ray data)
How do the amount of DNA per cell and the number of sets of chromosomes per cell relate?
by a precise correlation
How does the molecular composition of DNA compare between all the different cells of an organism?
it’s the same
How does the composition of both RNA and proteins compare between different cell types?
highly variable
How does the stability of DNA and RNA/proteins compare?
DNA is more stable, while RNA and proteins are synthesized and degraded quite rapidly in living organisms
Why is DNA more stable than RNA?
it’s double-stranded and lacks an OH group that could be acted upon
What were the three possible models for DNA replication?
conservative, semiconservative, and dispersive
What does the conservative model of DNA replication predict the products of the first and second replication will be?
first replication: one DNA molecule of both template strands; one of both copied strands
second replication: 3 molecules of all copied strands; 1 molecule of original template strands
What does the semiconservative molel predict the products of the 1st and 2nd replication will be?
1st: 2 molecules each of 1 template and 1 copied strand
2nd: 2 molecules with 1 parent and 1 copied strand; 2 with just copied strands
What does the dispersive model predict the products of the 1st and 2nd replication to be?
1st: 2 molecules with bits of template in each strand
2nd: 4 of the same
What experimental procedure was used to show that the semiconservative model was correct?
bacteria were cultured in a medium with a heavy N isotope, then transferred to a medium with a lighter isotope; the DNA sample was then centrifuged after the 1st and 2nd replications
How did centrifuging the products from the 1st and 2nd replications from the bacteria DNA replication experiment support the semiconservative model?
more dense DNA settled to the bottom while less dense settled on top; the results only matched the semiconservative model
What is the origin of repliaction?
where DNA replication starts on a chromosome
How many origins of replication are found on a circular choromosome?
1
How many origins of replication are found in a eukaryotic chromosome
many
Why can 1 strand of DNA serve as a remplate for a new one?
the 2 strands are complimentary
Why do circular chromosomes not have the same problems with replication as eukaryotes?
eukaryotic chromosomes are linear, so it’s not possible to replicate all strands in 5’ to 3’ direction (whereas in circular, can just continue around molecule in correct direction until it’s all done)
also, linear chromosomes end up losing some of their end each time (primers added)
How is the leading strand synthesized?
continuously, moving toward the replication fork
How is the lagging strand synthesized?
as a series of segments (okazaki fragments)
Why is a primer required for repliacion?
DNA polymerases can’t initiate synthesis (on a single strand) - need something to build on
What is a primer?
short stretch of RNA, synthesized by primase, using parental DNA strand as template
In what direction can a new strand elongate in replication and why?
5’ to 3’ since polymerase can only add nucleotides to free 3’ end of growing strand
What does helicase do?
enzyme that untwists double helix at replication fork
What do single-strand binding proteins do?
bind/stabilize by replication fork during replication - prevent retwisting
What is another name for topoisomerase?
gyrase
What does topoisomerase do in replication?
corects “overwinding” ahead of forks by breaking, swiveling, and rejoining DNA strands
What does primase do in replacation?
makes the primer
How many RNA primers are needed to synthesize the leading tsrand?
1
What happens after the lagging strand is synthesized in fragements?
DNA polymerase I replaces RNA primer with DNA; DNA ligase joins sugar phosphate backbones of fragments into continuous strand
How long is each okazaki fragment?
1000-2000 nucleotides long
How many RNA primers does the lagging strand need?
one for every Okazaki fragment
What model does DNA polymerase follow to synthesize DNA/
replication machine - “reels” in parental DNA and “extrudes” newly-made daughter DNA molecules (assisted by other proteins)
What occurs immediately after DNA synthesis?
proofreading
WHat enzyme proofreads DNA?
DNA polymerase
What can happen in DNA polymerase misses mistakes?
- mismatch repair (other enzymes correct)
What kind of mistakes can be corrected in DNA proofreading?
DNA damage by expoure to harmful chemical or physical agents or other spontaenous changes
What is nucleotide excision repair?
nuclease cuts out and replaces damaged stretch of DNA
What does DNA ligase do?
rebuilds the sugar-phosphate backbone of DNA whenever DNA is cut and nucleotides are replaced
What is the error rate after prroofreading?
Low but not zero
What happens when DNA sequence changes become permanent?
can be passed onto next generation (mutations)
What are the source of genetic variation upon which natural selection operates?
mutations (changes in DNA sequence)
Why do DNA molecules become progressively shorter after each replication?
can’t complete the 5’ ends, so repeated rounds of replication produce shorter DNA with uneven ends
Why isn’t progressive shortening a problem for prokaryotes?
have circular chromosomes
What is a codon?
3 nucleotide “word” that codes for an amino aid
What is transcribed into complementary, non-verlapping codons of mRNA?
codons of a gene
What happens to mRNA codons?
translated into a chain of amino acids, forming polypeptide
What happens if codons are not read in the correct reading frame?
frameshift mutation
What is the correct reading frame?
correct groupings of nucleotides necessary for the specified polypeptide to be proudced
What are telomeres?
special nucleotide sequences at ends of eukaryotic chromsomes (TTAGGG repeated 100-10000 times in humans)
What is the function of telomeres?
postpone the erosion of genes near the ends; may protect cells from cancerous growth by limiting number of cell divisions
What is connected to aging?
shortening of telomeres?
What catalyzes the lengthening of telomeres in germ cellls?
telomerase
What is there evidence of activity of in cancer cells?
telomerases
What is the central dogma of molecular biology?
concept that cells are governed by sequential, one-way chain of command (DNA –> RNA –> protein)
What is encoded into DNA by triplet code?
instructions for assembling amino acids into proteins
How many total codons are there?
64
How many codons code for amino acids?
61
How many “stop” signal codons are there, and what do they do?
3; signal to end translation
Why is the code redundant?
more than one codon may specify a particular amino acid
Wh is the code not ambiguous?
no codon specifies more than one amino acid
What is transcription?
the syntehsis of an RNA strand using info in DNA
What is produced by transcription?
mRNA (messenger RNA) - the transcript
What is translation?
the synthesis of a polypeptide, using info in the mRNA
Where does translation occur?
on ribosomes
What two things does translation requrie?
rRNA (ribosomal RNA) on ribosomes; tRNA (transfer RNA)
Is the template strand the same strend for a given gene?
yes, always
What is transcription unit?
the stretch of DNA that is transcribed?
What is the promoter?
DNA sequence where RNA polymerase attaches to start transcription
What is the terminator?
the sequnce that signals the end of transcription (ONLY in bacteria)
What are the 3 stages of transcription?
initiation, elongation, termination
What happens in the initian stage of transcription?
promoters signal transcriptional start point on DNA, transcription factors mediate binding of RNA polymerase and initiation of transcription
What is a transcription initiation complex?
the completed assembly of transcription factors and RNA polymerase II bound to a promoter
What is the TATA box and what is it crucial for?
part of promoter; crucial in forming intation complex in eukaryotes
Why is the TATA box a good target site for inhibiting transcription?
it’s necessary ton initiate transcription - stick chemicals there to stop binding
What is the role of transcription factors?
mediate the binding of RNA polymeraase and the initiation of transcription - ensure bonding, increase promoter binding
What would happen if transcription factors were note present (or were removed on purpose)?
reduction in efficiency of trasncription
What happens in the elongation phase of transcription?
RNA polymerase moves along DNA, untwisting double helix and transcriping genes
At what point of the growing RNA molecule are nucleotides added?
3’ end
What is the rate of transcription in eukaryotes?
40 nucleotides per second
How is transcription terminated in bacteria?
polymerase stops transcription at end of terminator site and mRNA is translated into protein without further modification
How is transcription terminated in eukaryotes?
RNA polymerase II transcribes the polyadenylation signal sequence (string of As) before being released
What postranscriptional modifications are done to mRNA?
- 5’ end gets modified nucleotide 5’ cap
- 3’ end gets poly-A-tail
What is the funciton of postranscriptional modifications?
faciliate export of mRNA to cytoplasm, protect it from hydrolytic enzyemes, help ribosomes attach to 5’ end
What are introns?
long noncoding stretches of nucelotides that lie between coding regions
What are exons?
sequences that are eventually expressed (usually translated into amino acid sequences)
What does RNA splicing do?
removes introns and joins exons, creating mRNA molecule with continuous coding sequence
What are spliceosomes?
complexes of proteins plus several small nuclear ribonucleoproteins (snRNPs) that recognize thesplice sites
What is a protein domain?
a discrete region of the modular architecture of a protein
What may code for different domains in a protein?
different exons
What may be the result of exon shuffling?
the evolution of new proteins
What is the functional and evolutionary importance of introns?
- some contain sequences that may regulate gene expression (rather than coding for protein)
- alternative RNA splicing
What is alternative RNA splicint?
some genes can encode more tahn one kind of polypeptide, depending on which segments are treated as exons during splicing
What adavantage does alternative RNA splicing give?
can produce more different proteins than there are genes
Where does protein synthesis occur?
on ribosomes
What 2 steps are required for accurate translation?
- correct match between transfer RNA and amino acid
- correct match between tRNA anticodon and mRNA codon
What is the anticodon?
region of tRNA of 3 bases gthat are complementary to a codon in mRNA
How long is a single tRNA strand?
80 nucleotides long (small)
What does the 2D structure of a tRNA molecule look like?
cloverleaf
What is the 3D structure of a tRNA molecule?
L-shaped molecule (due to hydrogen bonds twisting and folding it)
Why are the ends of tRNA molecules not identical?
one end binds to specific amino acid; other has anticodon that corresponds to that amino acid
What does a tRNA anticodon base pair with?
complementary codon on mRNA
What is wobble?
flexible pairing at the 3rd base of a codon to allow some tRNAs to bind to more than one codon
Why is a mutation within a wobble base not necessarily have an affect?
codons can have interchangeable nucleotides - if some are switched, may still code for same amino acid
What does the enzyme aminoacyl-tRNA synthetase do?
ensures a corect match by joining a specific amino acid to a specific tRNA (requires energy from ATP)
What are the 2 ribosomal subunits (large ans small) made of?
proteins and ribosomal RNA (rRNA)
WHat do ribosomes do?
faciliate specific coupling of tRNA anticodons with mRNA codons in protein synthesis
How many sides where tRNAs can attach to ribosomes contain, and what are they called?
3; E, P, A
What ribosomal site do polypeptides grow out of?
P site
What is the A site in a ribosome?
initial attachment site - holds tRNA that carries next amino acid to be added to the chain
What is the P site in a ribosome?
the central site - holds the tRNA that carries the growing polypeptide chain
What is the E site in ribosome?
exit site - where discharged tRNAs leave the ribosome
What are the two ends of a protein?
carboxyl and N-terminus
What do free ribosomes do and where are they located?
in cytosol; mostly synteheize proteins that function in hte cytosol
What do bound ribosomes do and where are thy found?
attached to the ER; a=make proteins of the endomembrane system and proteins secreted from the cell
Where does polypeptide syntehsis always beigin?
in cytosol
What is the exception to polypeptide syntehsis always finishing in the cytosol?
pollyypeptide signals the ribosome to attach to the ER
What is a polyribosome (Polysome)?
a single mRNA translated continuously by many ribosomes
What do polyribosomes allow cells to do?
make many copies of a polypeptide very queickly
In what types of cells are polyribosomes found?
both bacteria and eukaryotic cells
What are the 3 stages of translation?
initiation, elongation, termination
What aid in all the translation stages?
protein factors
Inw hat direction are mRNA base tirplets (codons) read?
5’ to 3’ direction
In what direction does translation proceed, and why?
5’ to 3’, since that’s the direction in which mRNA codons are read
What does each codon specify?
the amino acid to be placed at the corersponing position along a polypeptide
How many amino acids are there?
20
What occurs in translation initiation?
- small ribosomal subunit binds with mRNA and initiator tRNA
- small subunit moves along mRNA until reaches start codon (AUG)
- initiation factors (proteins) bring in large ribosomal subunit aht copmletes the translation initiation complex
What is the start codon?
AUG
What occurs during translation elongation?
amino acids are added one by one to preceding amino acid at the C-terminaus of the growing polypeptide chain
What are the 3 steps of elongation in translation?
- codon recognition
- peptide bond formation at C-terminus
- transloacation
What does elongation in translation require?
energy and proteins (elongation factors)
What are the steps of trnalstion termination?
- stop codon in mRNA reaches A site
- A site accepts protein called release factor
- release factor causes addition of watermmoleceule instead of amino acid
- reaction releases polypeptide, and translation assembly comes apart
Why is a functional protein not immediately released from translation?
translation makes a polypeptide, which often requires more work to become functional protein
What helps a polypeptide chain fold correcltyl?
chaperone protein (chaperonin)
What might polypeptides require before being functional proteins?
post-translational modifications
What are some post-translational modications?
- sugars, lipids, or phosphate groups added to amino acids
- one or more amino acids removed from amino end
- some polypeptides activated by enzymes that cleave them
- other polypeptides combine to form subunits of protein
- some polypeptides targetted for ER
How are protins targetted to the ER?
polypeptide gets marked by attaching signal peptide; signal-recognition particle (SRP) binds to signal peptide; SRP brings the signal peptide and its ribosome to the ER
What is the role of a signal-recognition particle (SRP)?
binds to singla peptide and brings it and its ribosome to the ER
Why are transcription and translation simpler in bacteria?
no nucleus - both processes can occur in same part of cell; protein syntehsis can begin as soon as transcript syntehsis begins
What are some causes of mutaitons?
spontaneous mutations from DNA replcation, recombination, or repair; physical/chemcial agents (mutagens)
What can mutations affect?
proteins sturcutre and function
What are point mutaitons?
small-scale chemical changes, in ust one base pair of gene
What are 2 point muation examples?
nucleotide-pair substitutions, nucleotide-pair insertions/deletions
What is an example of a disease caused by a poin mutaiton?
sickle cell disease (1 change of amino acid)
What does a nucleotide-pair substitution do?
replaces one nucleotide (and its partner) with another pair of nucleotides
What are silent mutations?
have no effect on amio acid produced by codon (due to redundancy in genetic code)
What are missense mutations?
still code for amino acid, but no the correct one (changes the protein)
What are nonsense mutaitons?
change an aminoa cid codon into a stop codong, nearly always leading to nonfunctional prtoein
What are insertions and deletions?
additions or losses of nucleotide pairs in a gene
Why do insertions and deletions often ahve a more disasterous efect on the resulting protein than substitutions?
may alter reading frame, causing farmeshift mutaiton - can mess up every subsequent amino acid
