Exam 3: DNA, Chromosomal Structure, Replication, Transcription, RNA Processing (Bio 375 - Genetics) Flashcards
Friedrich Meischer (1868)
doctor who isolated nuclei from pus cells (white blood cells + bacteria cells), chemically extracted nuclein [acidic and high in phosphorous … renamed to nucleic acid] (which was also found in nucleus of other cell types)
James Watson and Francis Crick
first to develop molecular model of DNA structure in 1953; used information from many researchers, including the X-ray crystallography recorded by Franklin and Wilkins and information about chemical composition
nucleic acid structure
composed of nucleotides
parts of a nucleotide
pentose sugar, phosphate group, nitrogenous base
pentose sugar
a five-carbon sugar molecule found in nucleic acids… deoxyribose (in DNA) contains no OH group on carbon 2’ and ribose (in RNA) contains an OH group on carbon 2’
nitrogenous bases
attached to 1’ carbon of pentose sugar; includes purine and pyrimidine components
purine
contains a double ring; includes Adenine (A) and Guanine (G)
pyrimidine
contains a single ring; includes Cytosine (C), Thymine (T) (found in DNA), and Uracil (U) (found in RNA)
chargaff’s rules
amount of adenine (A) is always equal to amount of thymine (T) and the amount of cytosine (C) is always equal to amount of guanine (G)
nucleoside
base + sugar (without phosphate group)
genome
entirety of genetic information
methylation
alters structure of base, leading to alteration of chromatin structure and inhibition of transcription; reversible process and often found in CpG islands (parts of genome with many CG pairings in a row) clustered in the genome
phosphate group
attached to 5’ carbon of pentose sugar; has a strong negative charge… can have up to three attached phosphates
dAMP, dADP, dATP
deoxyribose Adenine Mono/Di/Tri Phosphate
DNA structure
nucleotides (base + sugar + phosphate) form polynucleotide strands linked via phosphate groups between adjacent nucleotides using phosphodiester bonds … directionality is 5’ to 3’
DNA structure
NUCLEOTIDES (base + sugar + phosphate) form polynucleotide strands linked via phosphate groups between adjacent nucleotides using PHOSPHODIESTER BONDS … directionality is 5’ to 3’ … two strands form a DOUBLE HELIX that is ANTIPARALLEL… strands linked using hydrogen bonds and stacked bases … strands are COMPLEMENTARY
specificity of hydrogen bonding between nitrogenous bases
A=T (two hydrogen bonds) and G≡C (three hydrogen bonds)
RNA structure
usually single stranded… can base pair with itself to form hairpins or a single strand of DNA
information flow in cell
- REPLICATION (genetic information to descendants; DNA -> DNA)… 2. TRANSCRIPTION (transfer information to RNA; DNA -> RNA)… 3. TRANSLATION (translate information into proteins (RNA -> proteins)
4 levels of polynucleotide structure
- Primary… 2. Secondary… 3. Tertiary… 4. Quaternary
primary polynucleotide structure
nucleotide sequence of a single strand
secondary polynucleotide structure
base paired strands
tertiary polynucleotide structure
double helix
quaternary polynucleotide structure
higher order folding into cellular spaces facilitated via polynucleotide-polynucleotide and polynucleotide-protein interactions
transposable elements
any DNA sequence capable of moving from one place to another within the genome
genomics
study of structure and function of genomes
Cas-CRISPR
modifies genes/gene editing
B-DNA
Right-handed helical structure of DNA that exists when water is abundant; tertiary structure that is the most common DNA structure in cells due to being the most stable conformation… 10 base pairs per turn
tertiary DNA structure
either A (most compressed/least extended, right handed), B (right handed, most stable conformation), or Z (least compressed/most extended, left handed) forms… has major and minor grooves
quaternary DNA structure
supercoiling of the DNA double helix
supercoiling
when DNA helix is subjected to rotational strain while ends of molecule are stabilized by proteins; shortens DNA
positive supercoiling
DNA is overrotated, so helix twists on itself
negative supercoiling
DNA is underrotated, so helix twists on itself in opposite direction
topoisomerases
enzymes that add/remove rotations from DNA helix by temporarily breaking nucleotide strands, rotating ends around each other, then rejoining broken ends
most native DNA is negatively supercoiled because
it denatures more easily and fits into tighter spaces
archaea have positive supercoiling
so their DNA does not denature as easily in the extreme environments they are living in
bacterial chromosomal strucutre
overall form is circular; NUCLEOID consisting of a series of twisted loops held by proteins
nucleoid
“clump” of bacterial DNA that is confined to a definite region of cytoplasm; consists of a series of twisted loops held by proteins
degree of chromatin packing varies
during the cell cycle (less condensed during interphase when the replication is occurring); locally during transcription and replication
types of chromatin
euchromatin, heterochromatin
chromatin
combination of eukaryotic DNA with protein
euchromatin
undergoes normal changes in condensation during cell cycle; comprises the majority of chromosomal material
heterochromatin
always remains in a highly condensed state (even during interphase); found in centromeres and telomeres, Barr bodies, and the Y chromosome
chromosomes are present
only during the M phase
chromatin is present
throughout the cell cycle
histones
most abundant proteins in chromosomes/chromatin… consists of five major types (H1, H2A, H2B, H3, H4)… contains a high percentage of lysine and arginine amino acids (which give histones a net positive charge that attracts negative charges on phosphates of DNA)
other proteins in chromosomes and chromatin
non-histone proteins; scaffold proteins (which help fold and pack chromosomes)
nucleosomes
simplest level of chromatin structure… components: octameric core of histone proteins (all histone proteins except H1) and 1.65 DNA wraps
chromatosomes
components: nucleosome + H1 histone (which locks DNA into histone core)… separated from one another by linker DNA (and/or nonhistone chromosomal DNA)
higher order chromatin structure
nucleosome/chromatosomes twist around one another to form a tightly packed 30 nm fiber… 30 nm fibers loop and fold to form 300 and 250 nm fibers with scaffold proteins anchoring the loops… in M phase, tight coiling of 250 nm fibers produces 700 nm chromatid
chromatin structure
double stranded helix… nucleosomes… chromatosomes… 30 nm fiber… 300 nm loops… 250 nm fiber… chromatid of chromosome
chromatin structure during transcription
chromatin is relaxed in active areas of transcription… acetylase enzymes reduce charge of histones which cause the histones to release their DNA and allowing for better transcription (transcription factors are permitted to bind to DNA)… deacetylation is stimulated by nucleotide methylation (where the methylation can activate or repress transcription depending on which amino acids are methylated)
chromatin-remodeling complexes
proteins that alter chromatin structure without altering chemical structure of histone directly… they bind directly to particular sites on DNA and reposition the nucleosomes to allow transcription factors to bind to promoters and initiate transcription
telomeres
natural ends of a chromosome; caps and protects end of eukaryotic chromosomes from degradation and aids replication of chromosomal ends… structure: single stranded overhang, loops around to base pair with itself, bound by telomere proteins
telomeric sequences
repeated units of a series of adenine or thymine nucleotides followed by several guanine nucleotides
replication
always proceeds 5’ to 3’… its mode depends on the structure of the template DNA (whether it is circular bacterial chromosome vs linear eukaryotic chromosome)
replication always proceeds
5’ to 3’
replicon
individual unit of replication; contains one origin of replication
semiconservative replication
original nucleotide strands remain intact; original DNA molecule is semi conserved during replication
theta replication
occurs in circular bacterial chromosomes; both strands are replicated… occurs in a bidirectional manner involving a replication bubble and a replication fork
replication bubble
a region of DNA, in front of the replication fork, where helicase has unwound the double helix
replication fork
A Y-shaped region on a replicating DNA molecule where new strands are growing
rolling circle replication
occurs in some viruses and bacterial F factor… one of the strands is broken (only one strand of parental DNA is replicated at a time)-> new nucleotides are added to 3’ end of broken strand-> 5’ end is displaced from circle… produces a single stranded DNA and a circular double stranded DNA… is unidirectional… the single stranded DNA can serve as a template for further replication events
linear eukaryotic replication
have numerous replicons that are each 20,000-300,000 base pairs in length… occurs slower than bacterial replication at a rate of about 500-5000 nucleotides per minute… is bidirectional
replication requirements
- single stranded DNA template… 2. dNTPs (deoxyribonucleoside triphosphate) to be assembled into a new nucleotide strand… 3. enzymes (DNA polymerase) that read the template and assemble the substrates into a new polynucleotide chain
DNA polymerases can only add nucleotides to free 3’OH end of a growing DNA strand, so replication occurs by:
- new DNA is synthesized from dNTPs… 2. 3’OH end of last nucleotide on strand attacks 5’ phosphate group of incoming dNTP… 3. two phosphates are cleaved off… 4. a phosphodiester bond forms between two nucleotides and phosphate ions are released
continuous replication
replication continues unhindered as DNA is unzipped
leading strand
one parental strand can be used as template for a continuous complimentary strand… undergoes continuous DNA synthesis
lagging strand
other parental strand that is copied away from the fork in short segments called Okazaki fragments… undergoes discontinuous DNA synthesis and entails more enzymatic activity
steps of prokaryotic replication
- initiation 2. Unwinding 3. Priming 4. Elongation 5. Termination
(steps of prokaryotic replication) Step One: initiation
single origin of replication (oriC); initiator proteins bind to oriC and cause a short section of the DNA to unwind to provide the single stranded DNA template (initiator proteins are present long enough to open the initial “bubble” of single stranded template)
(steps of prokaryotic replication) Step Two: Unwinding
DNA helicase, single strand binding proteins, and DNA gyrase help to expand the single stranded template for greater accessibility by simultaneously unzipping DNA
DNA helicase
breaks hydrogen bonds and moves down lagging strand 5’ -> 3’… moving the replication fork… negatively supercoiling to unwind DNA
single strand binding proteins
stabilize single stranded DNA by preventing it from reverting back to double stranded DNA
DNA gyrase
relieves positive supercoiling ahead of replication fork
(steps of prokaryotic replication) Step Three: Priming
DNA polymerase requires an existing 3’OH group to begin DNA synthesis… Primase synthesizes a short stretch of nucleotides called a primer (10-12 RNA nucleotides), with one primer per leading strand and a new primer for each Okazaki fragment
(steps of prokaryotic replication) Step Four: Elongation
DNA polymerase III and DNA polymerase I aid in extending the new strands of DNA, with DNA ligase finalizing the new strands
DNA polymerase III
adds new nucleotides in 5’ -> 3’ direction (polymerase) and proofreads 3’ -> 5’ (exonuclease)… is stalled if an incorrect nucleotide is added, so it will back in a 3’ -> 5’ direction to replace the incorrect nucleotide with a correct nucleotide
DNA polymerase I
follows DNA polymerase III down the strand, removing RNA primers (exonuclease) and replacing RNA with DNA… however, it leaves “nicks” in the DNA strand
DNA ligase
final enzyme in replicative process… binds to nicks in phosphate backbone that remain after DNA polymerase I replaces RNA primer… seals nicks in backbone by joining adjacent nucleotides with a phosphodiester bond
(steps of prokaryotic replication) Step Five: Termination
termination protein binds to a terminator sequence and blocks helicase action
replication occurs simultaneously on
leading and lagging strands
eukaryotic replication complexities
complex polymerases; chromatin structure (nucleosomes assembly occurring immediately after DNA replication); linear chromosomes (telomeres– physical ends to chromosomes)
telomerase
an enzyme containing an RNA template that is used to extend the single stranded DNA overhang of a telomere…. the extended single stranded DNA overhang is looped into a hairpin (has nonconventional base pairing) and used a primer (because there is a free 3’OH)
telomere age
the length of telomeres indicates “biological age” with a longer telomere indicating a younger biological age… only “immortal” cells (bone marrow, germ cells, intestinal cells) have telomerase which allows for telomere elongation
RNA
can form complex double stranded secondary structures (anti-parallel, hairpin loop); wider variation in structure and function than DNA
primary types of RNA
messenger (mRNA), transfer (tRNA), ribosomal (rRNA)
messenger RNA (mRNA)
carries genetic information from DNA to ribosome
transfer RNA (tRNA)
small RNA that contains a binding site for an amino acid
ribosomal RNA (rRNA)
part of ribosomal structure, site of protein assembly
primary types of RNA are produced using the process of
transcription
all eukaryotic RNAs are transcribed in the
nucleus
transcription unit
stretch of DNA that codes for an RNA molecule and sequences needed for transcription; contains promoter, RNA coding sequence, and a terminator
template strand
nucleotide strand used for transcription
nontemplate strand
nucleotide strand which is not used for transcription
the template and nontemplate strand are
complementary
the template strand and RNA transcript are
complementary
the nontemplate strand and RNA transcript are
identical (aside from T in nontemplate and U in RNA)
template strand is also known as
noncoding strand, antisense strand, minus strand
nontemplate strand is also known as
coding strand, sense strand, plus strand [because it shares the same sequence of nucleotides as RNA transcript]
making RNA from template
5’ -> 3’
reading template
3’ -> 5’
promoter
DNA sequence that transcription apparatus recognizes and binds
RNA coding region
sequence of DNA nucleotides that is transcribed to RNA molecule
terminator
DNA sequence that signals where transcription should end
upstream
direction towards the promoter
downstream
direction towards the terminator
transcription apparatus binds to the promoter and moves downstream towards the
terminator
transcription requirements
single stranded DNA template… ribonucleotide triphosphates (rNTPs) to be assembled into a new RNA strand… transcription apparatus (with RNA polymerase)
RNA polymerase
synthesizes a new RNA in a 5’ -> 3’ direction; the new RNA is complementary and antiparallel to template strand… does NOT NEED A PRIMER
DNA polymerase requires a
primer
bacterial RNA polymerase
large multimeric enzyme… holoenzyme composition: two alpha (α), one beta (β), one beta prime (β’), one omega (ω) [helps with stabilization], and one sigma (σ) [controls binding of polymerase to promoter]
core polymerase
composition: two alpha (α), one beta (β), one beta prime (β’), one omega (ω)…. responsible for extending RNA chain
holoenzyme
composition: two alpha (α), one beta (β), one beta prime (β’), one omega (ω) [helps with stabilization], and one sigma (σ)… only present at initiation, with the sigma (σ) required for specificity (different σ factors bind to a specific promoter of transcription unit)
eukaryotic RNA polymerase types
RNA polymerase I (transcribes large rRNAs)… RNA polymerase II (transcribes pre-mRNA, some snRNAs, snoRNAs, some miRNAs)… RNA polymerase III (transcribes tRNAs, small rRNAs, some snRNAs, and some miRNAs)… RNA polymerase IV (transcribes some siRNAs in plants)
prokaryotic transcription steps
initiation -> elongation -> termination
transcription step 1. initiation
σ factor associates with core polymerase to form holoenzyme (σ factor is required to direct holoenzyme to specific promoters) –>
holoenzyme binds to consensus sequence (-10 and -35) –>
holoenzyme unwinds and melts double stranded DNA from -10 start site –>
active site of holoenzyme aligns with start site –>
9-12 complementary RNA nucleotides (abortive transcripts) are added in a 5’ -> 3’ direction –>
σ is released, transforming the holoenzyme into the core enzyme –>
the core enzyme is released from the promoter and able to elongate the RNA transcript
consensus sequence
sequences that show considerable similarity between genes; includes the -35 sequence (TTGACA) and -10 sequence (TATAAT); is recognized by the holoenzyme during initiation
transcription step 2. elongation
RNA polymerase unwinds DNA as transcription progresses (making RNA), with the transcription bubble being about 18 nucleotides in length
transcription step 3. termination
core polymerase continues transcription until it transcribes the terminator –> termination occurs through either Rho-dependent termination or Rho-independent termination
Rho dependent termination
Rho protein binds RNA and moves 5’ -> 3’ down RNA –> upon reaching the terminator, RNA transcript forms a hairpin loop that pauses the RNA polymerase –> when Rho reaches RNA polymerase, it stops transcription by unwinding the RNA away from the DNA template strand
Rho
has helicase action that allows unwinding of RNA transcript from DNA template
Rho independent termination
upon reaching the inverted repeats present in the terminator, the RNA transcript forms into a hairpin loop, forcing the RNA polymerase to pause –> the relatively weak A-U hydrogen bonds break -> RNA separates from template and the polymerase is released
RNA polymerase does not need
primers or helicase
DNA polymerase needs
primers and helicase/gyrase
eukaryotic transcription differences
multiple promoters (core promoter, regulatory promoter)… more consensus sequences (-35 , -25 (TATA box), +1, +30…)… more complex RNA polymerase complex (transcription factors required for polymerase binding that correlate with all sequences of core promoter and regulatory promoter)… enhancers and silencers
regulatory promoter
controls promoters downstream; is upstream of the core promoter
enhancers and silencers
sequences distant from transcribed gene that can stimulate or repress transcription (respectively)
mRNA structure
5’ untranslated region; protein coding region; 3’ untranslated region
5’ untranslated region (UTR)
upstream of start codon; does not code for amino acids; Shine-Dalgarno rRNA binding site
protein coding region
between the start and stop codon
3’ untranslated region (UTR)
downstream of start codon and stop codon; does not code for amino acids; affects mRNA stability and translation
mRNA processing in prokaryotes
not extensively processed before translation; transcription and translation occur simultaneously
mRNA processing in eukaryotes
processed before translation; transcription (in nucleus) and translation (in cytoplasm) occur separately due to locations; initial mRNA produced from transcription is pre-mRNA and becomes mature following processing
prokaryotic gene structure
genes and proteins are collinear (no “breaks” in sequence from DNA -> mRNA -> protein)… direct correspondence between nucleotide sequence of a gene and amino acid sequence of a protein
eukaryotic gene structure
split genes:: contain exons (coding regions) and introns (noncoding regions removed from RNA using RNA splicing)
mRNA processing steps
- 5’ cap addition –> 2. Poly A Tail addition –> 3. mRNA splicing
mRNA processing step 1. 5’ cap addition
methylated guanine nucleotide is added
mRNA processing step 2. Poly-A Tail Addition
process: recognition of consensus sequence in 3’-UTR by tailing enzymes -> cleavage of pre-mRNA 11-30 nucleotides downstream of consensus sequence -> addition of 50-250 adenine nucleotides
5’ cap end
functions: facilitates ribosome bonding (differentiated shape directs binding of ribosome to capped end) which then aids in translation initiation, increases stability (prevents degradation), and influences splicing
poly-A tail
consists of 50-250 adenine nucleotides added to 3’ end of pre-mRNA… functions: increases stability of mRNA (prevents degradation), facilitates ribosome attachment, allows passage to cytoplasm (allows exit from nucleus)
mRNA processing step 3. mRNA splicing
process: 5’ splice site is cut by spliceosome -> 5’ end of intron attaches to branch point -> 3’ splice site is cut by spliceosome -> intron is released as a lariat -> exons are ligated
mRNA splicing
removal of introns from pre-mRNA… requires a 5’ splice site, 3’ splice site, and a branch point… process occurs in the spliceosome
spliceosome
one of the largest molecular complexes in eukaryotic cell; composed of 5 snRNAs and 300 proteins; snRNAs combined with proteins to form snRNPs (small nuclear ribonucleoparticles)
alternative mRNA processing
alternative splicing and multiple 3’ cleavage sites – ways to get varying mRNAs from one gene to make many different proteins
