Lecture 6 - From DNA to Proteins Flashcards
what does an ames test show?
it is a standard test in biotechnology that reveals mutagenic potential of the compounds by reverting histidine-auxotrophic phenotype of S. typhimurium
what are some examples of exogenous sources?
radiation, chemicals
what are some examples of endogenous sources?
replication errors, spontaneous hydrolysis
DNA damage may result in what?
mutations in somatic cells and germline cells
what are the types of DNA damage?
- oxidation (most frequent source of DNA damage)
- alkylation (addition of alkyl groups to bases)
- deamination (loss and/or substitution of amino groups at the bases)
- depurination/depyrimidination of bases
- formation of base dimers & more complex heterocycles induced by ionizing radiation and carcinogens
- single & double stranded DNA breaks
- mismatch (replication error)
what is deamination?
it is when cytosine is converted to uracil
what happens during depurination?
removes guanine or adenine from DNA
what is radiation a significant source of?
DNA damage that frequently causes various DNA modifications depending on the radiation energy
what are examples of ionizing radiation?
X-rays, UV light, and gamma rays
how does ionizing radiation cause significantly more damage?
in the form of ring openings, it causes fragmentation of bases and breakage of phosphodiester bonds (i.e. single and double stranded breaks)
how does UV light affect the DNA bases?
it induces the condensation of 2 ethylene groups into cyclobutane ring. which can occur in the cell between 2 adjacent pyrimidine bases (typically thymines)
what happens during alkylation of DNA?
adds a methyl group to guanine to yield O6-methylguanine
what is mistaken DNA alkylation caused by?
- alkylating agents normally present in the cell (such as S-adenosyl-methionine = a donor of methyl group for many intracellular reactions)
- toxins called alkylating agents such as nitrogen mustard
what does O6-methylguanine base-pair with?
CANNOT pair with cytosine, must pair with THYMINE
what does the formation of O6-methylguanine result in?
inherited mutation
what is the most frequent source of mutagenic alterations in DNA?
DNA oxidative damage
what are reactive oxygen species (ROS) and how do they arise?
ROS such as hydrogen peroxide, hydroxyl and superoxide radicals; they arise during ionizing irradiation and as byproducts of oxidative metabolism
what is the most detected product of DNA oxidation?
8-oxo-2’-deoxyguanosince (8-oxo-G)
what is used to measure the oxidative stress in cells and tissues?
accumulation of 8-oxo-G
what are the types of DNA repair?
- base excision repair
- nucleotide excision repair
- mismatch repair
- homologous recombination
- non-homologous end joining
how does methylation of DNA work?
specific methylases distinguishes “old” strand from the “new” one
what do proteins of mismatch repair complex recognize?
recognize unpaired (‘melted”) part of double-stranded DNA as both nucleotides are ‘natural’
what is the driving force of evolution?
DNA mutagenesis
the DNA repair system has not evolved to protect what?
the individuals after reproductive age
what are conserved or invariant nucleotide sequences?
those that encode for important genes or traits preserved in evolution
which type of sequences often indicate regions that encode for obsolete functions?
variable sequences
what is the evolutionary clock?
number and type of nucleotide substitutions accumulated over time in variable sequences
what was used as an original model system to study gene expression and its regulation?
bacteriophages
what do gene readout start with?
the synthesis of single-stranded RNA copy using double-stranded DNA as a template
the amount of genetic information released from each gene may be significantly amplified thru what?
thru increased rates of transcription and/or translation
what direction does the extension of the RNA strand go during synthesis?
5’ to 3’ direction; the 3’ end extends
during transcription, which RNA strand is complementary to one of DNA strands?
the nascent RNA strand
what is the coding strand?
the opposite to the template strand in double-stranded DNA helix
if the coding strand of DNA is:
5’ ATTGGTACTCC 3’
what would be the template strand and what would be the RNA strand?
template strand:
3’ TAACCATGAGG 5’
RNA strand:
5’ AUUGGUACUCC 3’
how are stem loops formed?
only in RNA since it is a single strand; the bases in the single strand seek stability by forming base pairs between bases on the same strand
how are RNA secondary structures stabilized?
by non-conventional (non-canonical) pairs, such as G-A and C-U
in order to synthesize RNA, what must RNA polymerase do?
it has to separate (melt) the strands first, making one strand available for readout
what direction does RNA polymerase move?
it moves along the double strand in the direction that the template strand is read from 3’ to 5’ direction
- this ensures that RNA as antiparallel strand is synthesized in 5’ to 3’ direction
describe the RNA-DNA hybrid
- thermodynamically more stable than the corresponding DNA-DNA hybrid in double-stranded DNA of the same size and sequence
- provides great stability to the moving transcription machinery that does not dissociate from DNA when it stops
what do messenger RNAs (mRNAs) do?
code for proteins
what so ribosomal proteins (rRNA) do?
forms the core of the ribosome’s structure and catalyze protein synthesis
what does microRNAs (miRNAs) do?
regulate gene expression
what do transfer RNAs (tRNAs) do?
serve as adaptors between mRNA and amino acids during protein synthesis
what are other noncoding RNAs do?
are used in RNA splicing, gene regulation, telomere maintenance, and many other processes
what is the paradox of transcription in prokaryotes?
the same enzyme should have high affinity to a particular DNA sequence in order to initiate transcription at a very specific place
- at the same time, the very same enzyme has to be tightly bound to the DNA during elongation of transcription regardless the sequence
how does the paradox of transcription in prokaryotes get solved?
by a special protein called sigma factor
does the sigma factor get released at first attempt? why or why not?
no, normally DNA polymerase will make many attempts to escape promoter by synthesizing short, abortive RNA products
what do sigma factors in bacteria cells allow?
allow switching transcription from housekeeping genes to a set of specialized genes using the same RNA polymerase core enzyme
what is the transcription terminator in prokaryotes?
a sequence that encodes a stem-loop in RNA followed by a polyU tract
bacteria has ___ RNA polymerase, while eukaryotes have a set of ___ nuclear enzymes?
one; 3
what does the basic eukaryotic transcription factor TBP (TATA-binding protein) insert?
inserts aromatic amino acid radicals (phenylalanines) between base pairs and changes its conformation so that the residues work like levers to bend (kink) DNA at the promoter
what are RNA-degrading enzymes (RNAses) for?
ensure that RNA copies are always fresh, and do not have unwanted modifications that may accumulate over time
what are the most important steps of eukaryotic mRNA processing?
capping, polyadenylation, and splicing
what is capping?
it is the covalent attachment of 7-methylguanosince to 5’ triphosphate
- starts right after the transcript emerges from RNA polymerase
what is the function of poly-A-polymerase?
it adds a poly-A-tail at the 3’ end in non-template manner
- before this addition, the 3’ untranslated region (3’-UTR) is trimmed
protein-encoding genes are interrupted by non-coding sequences called what?
introns
what are exons?
the coding regions in genes
what does splicing involve?
immature, newly synthesized eukaryotic mRNA gets the introns out and the exons fused (spliced) together
some introns require assistance from what?
small nuclear RNA (snRNA) protein complexes (snRNPs or “snurps”)
what greatly increases the variety of products that can be obtained from the same gene?
alternative splicing
what are the differences in how prokaryotes and eukaryotes handle their mRNAs?
- all steps of mRNA processing in eukaryotes occur simultaneously and start as soon as the corresponding parts of mRNA emerge from the transcription complex
- translation of bacterial mRNAs may occur simultaneously with their synthesis, as long as the 5’ end emerges
what is a codon?
a sequence of 3 nucleotides (triplets of nucleotides) from 5’ to 3’ end that encode for amino acid
what is degeneracy of genetic code?
a result of simple mathematical probability to have just sufficient code variations for each amino acid out of 20
- also provides additional protection from mutations
what are the designated codons that translate for start and stop sites?
- ALWAYS starts with AUG (methionine)
- stop codons do not encode for any amino acids; synthesis of polypeptide chain is interrupted once it hits any of the 3 stop codons
what is the open reading frame (ORF)?
linear sequence of codons that encode the polypeptide sequence, from the start codon to the termination codon
which 2 non-coding RNA types are the most important?
tRNAs and rRNAs
what is an anticodon?
a triplet of nucleotides that is complementary to RNA codon in antiparallel orientation
what is aminoacyl tRNA synthetase (transferase)?
enzyme that covalently attaches correct amino acid to the 3’ end of corresponding tRNA
what does aminoacyl-tRNA synthase create for translation?
aminoacyl-tRNA adapter
the energy of ATP hydrolysis is used to create what?
the high-energy bond between acyl group of amino acid and tRNA which is later used as an energy source for the synthesis of polypeptide bonds (aka translation)
where does the translation of all eukaryotic mRNAs occur?
in the cytosol
where does the synthesis of rRNA, trimming of rRNA, and assembly of large and small ribosomal subunits occur?
in the nucleoli
how many sites does a ribosome have that permits the recognition of RNA template and tRNA binding?
- E site = exit
- P site = peptidyl-tRNA
- A site = aminoacyl-tRNA
what happens during the initiation of translation?
- requires protein translation initiation factors that recognize a ribosome binding site (RBS), which is a specific nucleotide sequence that includes initiation codon ATG
what is the ribosome binding site (RBS) called in eukaryotes?
Kozak consensus
what is the RBS called in prokaryotes?
Shine-Dalgarno
what causes the termination of translation?
- protein release factor binds termination codon
- promotes dissociation of ribosomes on 2 subunits that are recycled
how is the termination of translation different in eukaryotes vs prokaryotes?
- in eukaryotes, a single mature mRNA encodes for a single polypeptide
- in prokaryotes, a single mRNA may encode several polypeptides with their own sites for translation initiation AND termination
what is ubiquitin?
a small protein that marks other soluble cytosolic proteins for degradation by proteasome
what is proteasome?
a large protein-degrading complex that utilize short-lived and misfolded proteins
what can reprogram any differentiated cell into a pluripotent cell that has embryonic stem cell-like properties?
artificial expression of a set of 4 genes (each of which encodes for a transcription regulator protein)
what may provide clues on how the development of late onset diseases may be triggered?
induced pluripotent stem cells (IPSCs)
how would patient-specific cells differentiated from IPSCs may be compared with similar cells?
they would be isolated from healthy subjects in regard to how they respond to stimuli, stress conditions, and treatment in vitro
what are the 3 different control regulation steps that happen during gene expression?
1) transcriptional control
2) mRNA degradation control
3) translation control
when is transcription control exerted?
at the level of transcription initiation; either permitting or preventing the binding of RNA polymerase at the promoter region
what is the leucine zipper?
a crisscrossed helical element that is found in a number of transcription regulators that is capable of accessing the DNA major groove along the double helix
- is so tightly structured that the distance between amino acid residues that interact w base pairs is strictly maintained
what roles do the zinc fingers and helix-turn-helix motifs perform?
orienting recognition elements so that they bind certain base pairs at the promoter DNA
what is another example of transcription activator?
homeodomains
what do many transcription regulators bind to DNA as?
dimers
in bacterial chromosomes, how are genes fixated and expressed?
clustered and often expressed together
what is an operon?
a set of genes expressed from a single promoter
- typical for BACTERIA
what are bacteria incapable of storing?
large amounts of polysaccharides
what is a constitutive promoter?
one that works all the time as long as RNA polymerase is available
what is a promoter that is inducible?
they are transcriptional switches that control operons to allow transcription from those promoters only at certain conditions
- so that the cell can respond to the changes in their environment very efficiently
what are operators?
regulatory elements that control the activity of promoters by suppression
where are operators located?
- either adjacent to the basic promoter sequence
- or overlaps with the promoter
how are bacteria capable of turning off corresponding anabolic genes?
by using allosterically regulated transcription repressors
what is a repressor?
a special regulatory protein
where do the promoter sequences located on DNA?
typically on -35 and -10
describe the example of the bacteria E. coli using transcriptional repressors
- E. coli can either synthesize tryptophan or uptake it from surroundings
- in presence of imported tryptophan, bacteria DO need enzymes to synthesize it & corresponding operon that encodes for the set of tryptophan-synthesized enzymes is turned OFF w/ a repressor
- repressor binds tryptophan in such a conformation locks the access of RNA polymerase
in the absence of imported tryptophan, what happens?
the repressor cannot bind to the operator DNA, and the block is removed
what are transcriptional activators/activator proteins for?
it recruits RNA polymerase to the promoter and ensures productive transcription
what does the Lac operon in E. coli control?
the set of genes that utilize lactose by breaking it down to galactose and glucose when it is available from the extracellular sources
what happens when glucose is available. versus lactose?
bacteria prefers glucose and will then turn off the transcription of lac operon off
what happens when there is an absence of glucose?
- bacteria makes cAMP (which allosterically activates CAP)
what is CAP?
the lac operon activator that activates a number of genes allowing bacteria to use various alternative sources (including lactose)
what happens if lactose is absent?
lac operon specific repressor binds operator and turns the operon off
what happens in the presence of lactose?
its byproduct allolactose binds repressor and removes the transcription block
what is the MOST complex molecular process in eukaryotes?
transcription regulation
what are enhancers?
distant DNA elements that recruit activator proteins and thus permit transcription
how is coordinated expression of certain genes achieved in eukaryotes?
by bringing together RNA loops from different parts of chromosomes
how is transcription usually activated in eukaryotes?
thru the loosening of chromatin
what are 2 things that can be inherited by the daughter chromosome?
- histone
2. DNA modifications
how can sequence-specific binding protein repress the translation of some mRNAs?
by keeping the ribosome from binding to the ribosome-binding sequence on mRNA through the stabilization of double-standard (stem-loop) structures that encompass ribosomal binding site (RBS)
- thus makes RBS inaccessible to ribosome assembly as it would require single-stranded unfolded mRNA
what do MicroRNAs (miRNAs) do in eukaryotic cells?
direct the destruction of target mRNA; regulate gene expression by targeting complementary RNA molecules for destruction`
what is silencing?
negative regulation of transcription that controls gene expression of the level of mRNA stability
why are small interference RNAs (siRNAs) produced?
they are produced in response to foreign double-stranded RNAs
where can double-stranded RNA (dsRNA) come from?
either normally of viral origin or from transposones
what are transposones?
mobile elements that can move from one part of the genome to another interfering with normal genome activity
what is artificial RNAi used for?
to downregulate (or silence) specific genes in cultured cells and model organisms
