Exam 3 Flashcards
RNA polymerase
- Holoenzyme that finds which gene to transcribe, and begins the process of transcription
- no primer is necessary to begin transcription
- has 4 subunits bound to a core enzyme
- Sigma Factor plays role in regulatory function
- 3 types to synthesize the 3 types of RNA.
- RNA polymerase synthesizes mRNA,
- DNA polymerase synthesizes DNA
Centromere
- region of a chromosome where spindle fibers attach
- chromosomes without a centromere would be lost
- important for chromosomal segregation
Chromatin Remodeling
- structure must change to allow access to DNA
- ability to package DNA while maintaining structure, and be able to go back in and change structure to access the DNA
- ESSENTIAL
- histone tails, acetylation, methylation and phosphorylation
DNA is a ________ and nucleotides are the building blocks of these molecules
- nucleic acid
Direction of B form DNA (watson and crick)
- 5’ to 3’ direction (Watson)
- 3’ to 5’ direction (crick)
- antiparallel to eachother
Ribozymes
- catalytic RNA (function as enzymes)
Elongation in bacterial transcription
- RNA polymerase
- unwinding of DNA ahead of transcription bubble and rewinding behind it - 5’ to 3’ extension of RNA
- DNA/RNA duplex within the bubble
How do you remove introns (noncoding regions)?
- Pre-mRNA undergoes splicing in the nucleus
- Requires 3 sequences:
1. a 5’ splice site
2. a 3’ splice site - a 5’ and 3’ splice site defined by GU, end in AG
3. branch site- 18 to 40 nucleotides upstream of 3’ splice site (Y-pyrimidine and R-Purine) - Splicesome is ESSENTIAL!!! It’s made of RNAs and proteins and it cuts out introns
G banding
- Stains condensed chromosomes to create a visual karyotype
- photographs the entire chromosome complement
Alternative Splicing
- yielding different transcripts from the same gene via mRNA processing
- moves around the exons to decide which ones will get expressed
- adds variation
Transposable sequences
- “jumping genes” that move around and insert themselves to cause mutations
- mobile and can potentially relocate within the genome
- make up a HUGE portion of the human genome
Prokaryotic DNA structure
- DNA without free ends can become supercoiled positive and negative
- most DNA are negatively supercoiled (with the help of topoisomerase)
- separation of strands is easier, occupies less space then relaxed
Difference between eukaryotic and bacterial DNA
- bacterial DNA is packed with proteins instead of histones
- are much smaller in size
- are often circular
What does insertion of the wrong nucleotide lead to?
- the incorrect positioning of 3’ OH which stalls polymerase
Polytene chromosome
- specialized chromosomes that do not exist in every organism
- Can be seen under microscope during interphase
many stranded chromosome - has undergone DNA replication without the separation of the replicated strands, which remain in exact parallel register
- large banded chromosomes that came about during chromosomal REPLICATION WITHOUT DIVISION
- Line up with each other at chromomeres (centers)
- have puffs at certain chromomeres
Do most somatic cells have telomerase activity?
NO
- we have a copy of the gene, but it’s usually turned off and inactive
- This means our telomeres are shortening w/ every replication as we age
Heteromeric Codons
- codons that have different ribonucleotides in them
- EX: ACA instead of just AAA
- We know that a codon is made of 2 A’s and 1 C, but we don’t know the exact order (exact codon)
- ACA, AAC, CAA are all possible
What is the eukaryotic origins for DNA replication
- ARS (autonomously replicating sequences)
- enable DNA to replicate
- there are many
Linear eukaryotic Replication
- eukaryotes
- has multiple origins(replicons)/ replication forks and bubbles
- do not break the nucleotide strand
- forms 2 new linear DNA strands
- bidirectional
Nucleoside
- consists of only a pentose sugar and nitrogenous base
- NO phosphate group
Denaturation of DNA
- MELTING DNA to pull strands apart by breaking hydrogen bonds between nitrogenous bases
RNA splicing
- Removes noncoding introns from pre-mRNA
- facilitates export of mRNA to cytoplasm
- Allows for multiple proteins to be produced
A-DNA form
- alpha helix (right handed/clockwise spiral)
- shorter/wider
- probably does not exist in nature
positive supercoil
- adding in rotations to circular DNA which causes supercoiling
-10 consensus sequence
- Pribnow Box
- where unwinding begins bc it’s A-T rich which have easier-to-break Hydrogen bonds
- TATAAT
- prokaryotes
what does speed of denaturation tell you?
- The faster the 2 DNA separates… the fewer hydrogen bonds had to be broken (there must’ve been more A’s and T’s and less G’s and C’s)
- More G’s and C’s means DNA will separate/denature slower
DNA replication Elongation Step
- DNA polymerases synthesize the DNA
- DNA polymerase 1 has 5’ to 3’ polymerase activity
- has 3’ to 5’ exonuclease activity (allowing correction of errors)
- has 5’ to 3’ exonuclease activity used to remove the primers
- main function is removal of primers
- DNA polymerase 3 has 5’ to 3’ polymerase activity
- has 3’ to 5’ exonuclease activity (allows correction of errors)
DNA polymerases function in DNA replication
- function in replication, recombination and repair
What does it mean that the reading frame of mRNA is nonoverlapping*?
- You read every 3 bases as 1 codon and continue
- No overlapping of the codons and no pausing in the reading
- AUA.CGA.GUC
Core promoter in eukaryotic transcription
- part of DNA that has a binding site for RNA polymerase II to bind to begin transcription
- includes TATA box (same thing as Pribnow box in prokaryotes)
- Region of DNA that facilitates attachment of RNA polymerase.
- Often includes region enriched in T and A (TATA box).
NOT transcribed. - Located upstream, located before transcription start site (5’ of synthesized RNA, 3’ of template DNA strand).
replication requirements
- ssDNA template (single strand)
- 2 dNTPs (deoxyribonucleoside triphosphate)
- discontinuous DNA synthesis
Circular Replication
- circular DNA (prokaryotes) do not have any ends
- so a 3’ OH groups is always available to keep replicating around the circle
Nucleosome Assembly
- chromatin assembly factors (CAFs) move with replication fork
Leading Strand
- continuous
- serves as the template for continuous DNA replication
- replicates TOWARDS the replication fork
- replicates in the opposite direction of the strand that goes in 3’ to 5’ direction
R banding
- A chromosome banding technique in which chromosomes are heated in a phosphate buffer
- Produces dark and light bands in patterns that are the reverse of those produced by G-banding
Hershey Chase Experiment conclusion
- demonstrated that DNA, not protein, was responsible for directing the reproduction of phage T2 during the infection of E. coli
- radioactive proteins did not enter the cell and was not transmitted to progeny, they only made up the coat on the virus
- radioactive nucleic acids entered the cell and was transmitted to progeny
CONCLUSION: PROVED DNA IS THE HEREDITARY COMPONENT, NOT PROTEINS
DNA gyrase
- moves AHEAD of replication fork making and resealing breaks in the double helical DNA to release the torque that builds up as a result of unwinding at the replication fork
- a type of topoisomerase, in prokaryotes only
- reduces torsional strain that builds as the fork moves
- prevents supercoiling
untwists DNA a little, seals it together, prevents too much twisting
Solution for telomere problems
- have an enzyme that replaces the ends (block this in cancer cells as therapy)
Rolling Circle Replication
- prokaryotes
- used by some viruses and F factor
- circular DNA
- has origin of replication
- unidirectional
- breaks nucleotide strand
- pull away the outer circle of chromosome and add in new strand
- forms one circular molecule and one linear molecule that may circulize
How do nucleotides attach to one another
- via phosphodiester bonds
DNA ligase
- joins the okazaki fragments b sealing nicks in the sugar phosphate backbone of newly synthesized DNA
- to fill the gaps in the lagging strand, much more active on lagging strand
- also works on leading strand
Central Dogma of molecular biology
- DNA (DNA replication)
- transcription
- RNA
- translation
- protein
- have to be able to replicate DNA
- be able to transcript DNA to RNA then translate to protein (which does functional work)
Acetylation
- neutralizes positive charge on histone protein, relaxes histone hold on the negatively charged DNA
- Found in euchromatic areas which explains why DNA there is active!
Basal Transcription Apparatus in Eukaryotic transcription
- Group of protein transcription factors that bind to promoter sites on DNA to start transcription
- TFIID binds to TATA box to position RNA pol. II correctly (make sure everything is in the right place for transcription)
- TAFs (TBP associated factors) combine with TBP to make TFIID
- Other factors bind to stabilize TBP/DNA:
Renaturation/Reassociation of DNA
- allows hydrogen bonds to reform between nitrogenous bases so the 2 strands of DNA join back together
what does speed of renaturation tell you?
- DNA doesn’t renature all at the same rate
- Highly repetitive - reattach quickly bc they just attach to any base that matches them
- Moderately repetitive - reattach kinda quickly
- Unique - reattach slowly bc they have to find their exact match that they separated from
What enzyme does RNA replication depend on?
- RNA replicase
TATA box in eukaryotic transcription
- DNA sequence in the promotor region of genes
- 30 nucleotides upstream from start site
- tells other molecules where transcription begins
- similar to the Pribnow Box
- found in most eukaryotes!
Terminator in transcription
- the site on a gene that tells RNA transcription to shut off
DNA structure has 3 levels to consider:
- Primary Structure- nucleotide sequence
- Secondary Structure- double stranded helix with nucleotides connected by phosphodiester bonds in a “sugar-phosphate” backbone connected to another strand by hydrogen bonds
- Tertiary structure- higher order packaging
ssDNA proteins
- prevent single stranded regions from snapping back
Eukaryotic Transcription vs Bacterial Transcription
- Eukaryotic cells have 3 RNA polymerases
- So, promoter definition depends on type of polymerase
- many proteins take part in binding of polymerase to DNA
- different proteins require different proteins - KEY- promoters are recognized by accessory proteins which recruit RNA polymerase
- in bacterial transcription holoenzyme recognizes the promoter
Intron
- Intergenic region
- Intervening sequence
- Part of pre-mRNA
Adenine is complementary with Thymine and forms _____ hydrogen bonds. Cytosine binds with Guanine and forms _____ hydrogen bonds
- 2
- 3
Solenoids
- compacted, coiled structure created by H1 Histones interacting and connecting Nucleosomes
- nucleosomes that wrap into coils
- Purpose? COMPACT THE DNA
Nucleotides three components
- Pentose sugar
- Phosphate
- Nitrogenous base
Codon
- three nucleotides that specify for one amino acid
3 mechanisms to prevent mistakes in DNA replication
1) DNA polymerases are very good at reading DNA template strand, & bringing in the correct complement base pairs - they usually pick the correct nucleotide
2) Proofreading ability of the polymerase in the 3’ to 5’ direction using an exonuclease to remove incorrectly inserted nucleotides
3) Fixing errors after replication via mismatch repair which checks the newly synthesized strand for errors and fixes them
Does gene size = protein size?
- No! because some regions are not transcribed, and some are transcribed but not translated
- due to introns and exons
Nirenberg and Matthaei
- Accidentally figured out the first codon
- Added polynucleotide Phosphorylase to a test tube with a lot of Uracils and ended up with a chain of amino acids
- Discovered that the U’s were being used as a template to translate into a protein chain of phenylalanine
- decided that 3 U’s = a codon that translates into Phenylalanine
- Then they repeated this with different bases (A’s, G’s, C’s, etc) to figure out which 3-base codons translated into which amino acids
- This only worked for AAA, CCC, and UUU bc there’s only 1 possible order for these sequences
Messenger RNA (mRNA)
- carries genetic code for proteins
- A copy of DNA is carried out of nucleus and used in translation to be turned into a protein
Dispersive Replication
- old chunk, new chunk, old chunk, new chunk, dispersed
- each daughter strand consists of both old and new DNA
- The original parental DNA is dispersed throughout the 2 replicated copies
- all mixys
DNA Primase
- form of RNA polymerase
- lays down RNA primers (10-12 nucleotides) for DNA polymerase III to extend
- allows for the initiation of DNA synthesis
- an RNA polymerase that synthesizes short oligonuclotide to get DNA replication started
- results in short fragments of RNA that provide suitable 3’ OH ends upon which DNA polymerase 3 can begin polymerization
Conservative replication
-The parent double strand is “photocopied” and maintained/conserved throughout several replications
- no hybrids
- the original helix is conserved
with a 3:1 ratio of new to old
Hershey Chase experiment
- provided evidence that DNA was the genetic material using E. coli and one of its infecting viruses, T2 bacteriophage
1) Labeled proteins to see if they’re transmitted to progeny
2) Radioactively label the sulfur protein coat on VIRUS
3) Make virus infect E. coli
4) Blend it to break virus apart from bacterial cells - centrifuge
5) heavier bacterial cells fall to bottom & light phages go to top
6) Saw that all radioactivity was at the top in the phages
7) Do the same with DNA using radioactive phosphorus instead of sulfur
8) Saw that all radioactivity was at the bottom in bacterial cells bc the radioactive DNA entered cells & was transmitted to progeny
Levene’s observation
- Came up with the TETRANUCLEOTIDE THEORY that DNA is made up of 4 bases in a fixed sequence,
- Identified the nucleotide (sugar, phosphate, & base) which is the basic unit of DNA
- proteins were initially favored as the genetic material because thought DNA is too regular to encode information
- Chargaff proved this to be incorrect when he demonstrated that most organisms do not contain equal amounts of nucleotides
Termination in bacterial transcription
- Rho independent termination
- Rho dependent termination
Difference in deoxyribose sugar and ribose sugar
- in deoxyribose there is an H group attached to 2’ carbon of sugar
- in ribose there is a OH(hydroxyl) group attached to 2’ carbon of the sugar
Taylor, Woods and Hughes
- showed that semiconservative replication occurred in eukaryotes
- Allowed replication to occur in unlabeled media, and labeled media
- Led to 1 labeled chromatin and 1 unlabeled chromatin
Holoenzyme
- a very large complex made of many components
- DNA polymerase III is an example because it has a bunch of different components (alpha, epsilon, beta, and more!)
- very important that replication works well, because this enzyme is so complex
- The active form of RNA polymerase
- Contains 4 subunits, sigma factor, and RNA polymerase. -
IN PROKARYOTES
Initiator proteins
- binds to origin and separates strands of DNA to initiate replication
Subunits to core enzymes in RNA polymerase
- 2x alpha (a)
- 1 beta (B)
- 1 beta prime (B’)
Eukaryotic transcription Termination
- RNA polymerase 1 requires a termination factor that binds to DNA downstream of termination site
- RNA polymerase 3 uses a termination sequence (string of Us in RNA molecule)
- requires no secondary structure - RNA polymerase 2 termination is coupled to cleavage in the 3’ UTR (untranslated region)
The poly A tail in eukaryotic mRNAs
- has 50 to 250 adenine nucleotides
- is not encoded by DNA
- added after transcription
- Long tail of Adenines added to the 3’ end of most eukaryotic mRNA’s to increase stability and prevent degradation
- Allows mRNA to leave nucleus and travel to cytoplasm to get translated into proteins by ribosomes
_______ bonds link nucleotides and help create the sugar-phosphate backbone
- phosphodiester bonds
rRNA
- abundance?
- function?
- once of the most abundant organelles in the cell
- makes ribosomes
Start codon (Met)
- AUG
DNA Pentose sugar
- deoxyribose
- contains H on 2’ carbon
SINE vs LINE
- Short interspersed element -
- Long interspersed elements
- types of transposable elements/jumping genes
If DNA synthesis occurs on both strands what is required?
- two DNA polymerase 3 are required
What serves as genetic material in some viruses?
- RNA
what happens to telomeres when we replicate? and what’s the consequence?
- they get shorter with each replication, so we will eventually lose them and thus lose the chromosome entirely
Promotor in transcription
- DNA sequence that transcriptional apparatus recognizes
- indicates which strand of DNA will be transcribed
- determines start site of transciption
- does not get transcribed itself
The human genome is composed of
- highly repetitive DNA and moderately repetitive DNA
- tandem repeats and interspersed retrotransposons: middle repetitive
- satellite DNA: highly repetitive
Structural features of tRNAs
- Cloverleaf secondary structure
- 4 major arms (acceptor, thymine pseudouracil cytosine, anticodon, dihydrouridine)
- 74-92 nucleotides long
Pyrimidines
- six member SINGLE ring
- cytosine, thymine and uracil
Transcription is a highly _____ process
- selective
- only bits of the genome are ever transcribed into RNA. not the entire genome
Transcription requires:
1. ssDNA template
2. Substrates to make RNA (Ribonucleotides triphosphate)
3. Transcription machinery (polymerase)
Franklin and Wilkins
- used X-ray crystallography to determine that DNA was some sort of helix
- was the key to unlocking DNA structure
- got a good picture of DNA by freezing it inside a crystal & shooting x rays at it
Consensus sequences of poly(A) tail
- AAUAAA
- Sequence downstream of cleavage site is
U/G rich
Methylation
- Methylate the histones and DNA to SHUT THEM DOWN
- Which is why it’s found often in heterochromatic areas (inactive DNA)
Origin of replication
- where DNA synthesis begins
Watson and Crick
- used chemistry and x-ray diffraction to solve DNA structure (franklins data was key!)
- purposed right handed double helix structure composed of two antiparallel polynucleotide chains held together by hydrogen bonds formed between complementary nitrogenous base pairs
- explained Chargaff’s rule A=T because adenine base bonds to thymine bases
DNA strands run in ______ directions
- opposite
- antiparallel
Template binding in transcription
- the initial step of transcription that occurs when the RNA polymerase sigma subunit recognizes specific DNA sequences called promoters
B form of DNA structure
- double stranded in a helix
- made up of polynucleotides in an antiparallel strand direction
TAFs (TBP associated factors) in Eukaryotic transcription
- combine with TBP to make TFIID
Anticodon
- An “arm” of tRNA that’s ESSENTIAL in translation
- matches up to the mRNA & brings in the correct amino acid
- binds to any codon that’s antiparallel and complementary
CEN region
- the smallest region on a centromere necessary for chromosome movement
- DNA region of chromosomes critical to their function
What is equal to 1/2 of proteins mass
- non histone chromosomal proteins (scaffolding proteins and DNA replication/maintenance proteins/transcription proteins)
Genetic code is degenerate
- amino acids may be encoded by more than one codon
Replication on fork requires (5)
- Helicase
- ssDNA
- DNA gyrase
- DNA primase
- DNA polymerase
Chromomeres
- areas where the polytene chromosomes all lined up
- the banding pattern is distinctive for each chromosome in a given species
- reveals differential gene activity
3’ end of DNA strand
- OH group
DNA Helicase function
- unwinds DNA at replication fork
- enzyme that breaks hydrogen bonds between the base of strands
- cannot initiate unwinding
- binds to the lagging strand template and moves 5’ to 3’ (moves the fork)
Lagging Strand
- discontinuous
- causes small fragments of new DNA strands to be synthesized with gaps between the fragments (Okazaki)
- at the end the gaps will be filled
- replicates AWAY from replication fork
- replicates in the opposite direction of the strand going in the 5’ to 3’ direction
-35 consensus sequence
- TTGACA
- 35 base pairs behind the +1 transcription start site on DNA
Exon
- coding region
- expressed
DNA Topoisomerase
- can relieve or introduce supercoiling
- in eukaryotes
- reduces the tensional strain caused as the fork moves
DNA must be accessible to __________
- RNA polymerases
- what gives with DNA that
Telomerase - structure & function & how does it carry out this function?
- Ribonucleoprotein that helps extend telomeres so they don’t get lost with lots of replications
- Can lead to cell longevity & potential cell “immortality” (cancer)
- Uses an RNA guide to carry out reverse transcription (turn RNA back into DNA)
Electrophoresis
- separates DNA fragments based on size
- Larger DNA pieces have more base pairs and are heavier so don’t travel far, and stay at top
- Possible bc DNA is negatively charged and is attracted to the positive end of the gel
RNA differences from DNA
- RNA contains uracil instead of thymine
- RNA is single stranded instead of double stranded, can aquire shape/folds (primary to secondary structure)
- Type of sugar is Ribose instead of deoxyribose
- presence of 2’ Carbon OH group instead of H group
- easily degraded instead of stable
- can be catalytic has ribozymes
Okazaki Fragments
- fragments caused by the discontinuous synthesis in the lagging strand
Genetic code characteristics
- Unambigous
- Degenerate
- Synonymous Codons
- Isoaccepting tRNAs
- Wobble
Theta replication
- Prokaryotes
- circular DNA
- Replication begins at origin of replication
- forms one replication bubble
- forms 2 replication forks at each end of bubble
- unidirectional OR bidirectional
- has 2 forks migrating in opposite directions toward eachother
- once the 2 forks meet two copies of circular DNA are formed
Rho independent termination in bacterial transcription
- Rho = specific terminator protein - this mechanism doesn’t need rhos
- terminates when inverted repeats form a hairpin followed by a string of uracils
1. contain inverted repeat sequences
2. Hair pin (stem loop) structure formation is required - this structure causes RNA polymerase to pause bc falls off strands
- also may destabilize RNA/DNA complex because theres a ton of As and Us which break easily
Chargaff’s Rule
- isolated DNA and found varied in composition
- A=T and G=C, but not equal amounts of both pairs
- Disproved the tetranucleotide theory by showing that DNA is not in a FIXED sequence
- Pyrimidines pair with purines
Rho dependent termination in bacterial transcription
- requires the protein Rho
- allows Rho protein to catch up with polymerase and knock it off the strands causing transcription to end
1. requires DNA sequence that causes a pause in transcription
2. stretch of DNA upstream of termination site that is devoid of secondary structure
The initiation of DNA synthesis
- A complementary RNA primer is synthesized to which DNA is added
- all synthesis in the 5’ to 3’ direction
- eventually the RNA is replaced by DNA via DNA polymerase 1
How many base pairs does a turn in the helix measure
- 10 base pairs
Regional centromere
- centromeres found in most plants and animals
- larger than point centromeres
Replication fork
- The Y shaped region of a chromosome associated with the site of DNA replication where the strands become unwound
- starts at the origin of synthesis when the strands of the helix are unwound
Genetic code is unambiguous
- one codon (or triplet) specifies only one amino acid
The 3’ end of most eukaryotic mRNAs are modified with.?
- the Poly A tail
proofreading of DNA synthesis
- synthesis is not perfect by DNA polymerase
- 3’ to 5’ exonuclease activity that allows to detect and delete inaccurate mismatched nucleotides
- once removed 5’ to 3’ synthesis will proceed
Transcription start site
- where transcription begins
B-DNA form
- the DNA that Watson and Crick solved
- most stable of DNA’s under physiological conditions, it is the happiest in this state
- exists in the presence of water
- alpha helix (right handed/clockwise spiral)
- 10 base pairs per rotation
Transcription
- transfer of genetic information from DNA by the synthesis of the complementary RNA molecule using a DNA template under direction of RNA polymerase
- Process of mRNA synthesis in the nucleus of eukaryotes from DNA (in cytoplasm of prokaryotes)
- shortening DNA to RNA to use the genetic information
- maintains the same “language” (still reads nucleic acids)
- the initial step in gene expression
- highly selective process
Eukaryotes have 2 types of rRNA genes
- Large one that encodes 18S, 28S and 5.8S
2. Other encodes 5S rRNA
Pseudogenes
- Genes that were once active but no longer are due to accumulated mutations
- Look like genes, but don’t actually function
Bacterial Transcription Initiation Step
- promotor recognition/binding
- key to determining frequency that a gene is transcribed - Formation of transcription bubble
- makes the first bonds between ribonucleotides
- Escape of sigma factor (it disassociates) from promoter
Bacterial RNA polymerase
- usually only one type, RNA polymerase 2
- synthesizes all classes of RNAS (mRNA, rRNA, rRNA)
5’ capping
- a modification of eukaryotic mRNAs
- Addition of a methyl group on the 2’ OH group of an extra added nucleotide at the 5’ end of pre-mRNA (before it’s mature)
- This creates a “cap” on the end of mRNA which protects it from degradation from nucleases when traveling into the cytoplasm…. and makes it mature so it can later undergo translation into proteins
SSBPs (single stranded binding proteins) function
- stabilize the open conformation
- attach to single stranded DNA and prevent secondary structures from forming
- after helicase splits the DNA strands, proteins bind to the strand to prevent it from binding back together
Where are the phosphate groups attached in B form DNA?
- between 5’ carbon of one sugar and the 3’ carbon of the adjacent sugar (left to right)
DNA replication Prokaryotic initiation step
- circular chromosome has one origin of replication
- the double helix unwinds, forming a replication fork at which synthesis begins
- initiator proteins bind to oriC, relaxing strand(gyrase), breaking H-bonds(helicase), and beginning replication
what 2 things are involved with keeping DNA unwound?
- ssDNA base pairs and topoisomerases
3 components to the Transcriptional Unit
- Promotor
- RNA coding sequence
- Terminator
3 major steps stages in bacterial transcription
- Initiation
- machinery assembles on promotors, begins synthesis of RNA - Elongation
- RNA polymerase reads DNA, adds ribonucleotide to growing RNA - Termination
- end of transcription, separation from DNA template
Nirenberg and Leder’s Triplet Binding Assay
- Made synthetic mRNAs of a known sequence of 3 bases
- Used to test the order of heteromeric codons & find codon for each amino acid (Ex: ACA, AAC, CAA)
- Amino acid to be tested was made radioactive, & a charged tRNA produced.
- Incubated together w/ ribosomes -> passed through nitrocellulose filter
- if radioactivity retained on filter, then charged tRNA bound to its correct triplet associated w/ ribosome
Result: Anticodon on tRNA binds to complementary codon on mRNA when connected to ribosome
Semiconservative Replication
- unfolds and make new copies of itself
- a SINGLE strand of DNA is conserved so that some hybrids are made, and some parental strands are kept (conserved)
2: 2 ratio of new and mixy
Phosphodiester Bond
- phosphate group links adjacent nucleotides extending from 5’ carbon of one pentose sugar and the 3’ carbon of the pentose sugar in the neighboring nucleotide
- phosphodiester bonds create the backbone of nucleic acid molecules
Retrotransposons
- RNA intermediates that use reverse transcriptase (turn RNA back to DNA)
TFIID in eukaryotic transcription
- Basal transcription apparatus
- protein that identifies the TATA box and binds to it to make sure that RNA Polymerase II is positioned correctly to start transcription
- Attracts RNA polymerase and other transcription factors so we can start transcription
- consists of about 9 proteins (including TBP)
How do splicesomes function?
- pre-mRNA is cut at 5’ splice site
- Intron folds back, making a lariat structure
- requires transesterification - pre-mRNA is then cut at the 3’ splice site to set the rest of the intron free and then it is gone!
euchromatin
- chromatin that can be transcriptionally active (open chromatin)
- openly packed
Scaffold proteins
- non histone proteins that play a role in folding and packing DNA
- The chromosomes here are the most tightly compacted
DNA replication Termination Step
- occurs when two forks meet
2. sequences in some systems bind termination protein that blocks helicase
Chromosome banding
- When you stain specific areas of chromosomes (often based on A=T/G=C composition)
- There is a fingerprint for each chromosome (unique to each one)
- Helps identify chromosomes
- Helps you see any changes or rearrangements within chromosomes or between diff chromosomes
- extremely accurate!
Messelson and Stahl
- evidence that semiconservative replication is the mode used by bacterial cells to produce new DNA molecules
- confirmed that semiconservative replication was the way DNA replicated
4 properties of genetic material
- replication
- storage of information
- expression of information to yield phenotype
- variation as a result of mutation
Calcitonin gene
- Example of altered 3’ cleavage site
Template strand in transcription
- the single stranded DNA or RNA molecule that specifies the sequence of a complementary nucleotide strand synthesized by DNA or RNA polymerase
- DNA strand used to make mRNA
- Complementary DNA strand called nontemplate/coding strand
Supercoiling
- as unwinding occurs a coiling tension is created ahead of the replication fork
- this tension often produces supercoiling
- in circular molecules supercoiling may take the form of added loops and turns in the DNA
- allows DNA to take up less space in the cell by coiling in on itself
- helps organize & compact it into the cell
purpose of chromosome structure
- allows very tight DNA packaging so the chromosome can fit in a cell
Ribosomal RNA (rRNA)
- structural and functional components of the ribosome
- doesn’t turn DNA into a protein, but is essential in making them
- Makes ribosomes, the “work bench”
- necessary to make EVERY protein
eukaryotic RNA polymerases
- these activities are the result of large multiprotein complexes
- RNA polymerases 1,2,3
Isoaccepting tRNAs
- different tRNAs carry the same amino acids
What are Progerias? and what are they associated with?
- Rapid aging disorders associated with shortened telomeres
- Hutchinson-Gilford syndrome
- telomeres shorten faster so the individual looks older
The cap is important for initiation of translation because
- Cap-binding proteins attach to cap, allowing ribosome to bind
- Cap increases transcript stability
- Cap influences splicing of introns
Transcription occurs where?
- inside the nucleus
Transfer RNA (tRNA)
- helps incorporate amino acids into polypeptide chain
- doesn’t turn DNA into a protein, but is essential in making them
- functions as a translator between nucleic acid and protein languages by carrying specific amino acids to the ribosomes where they reorganize
What happens if there is a mismatch pair?
- will fix errors after replication
DNA polymerase 3 in prokaryotes
- cannot initiate DNA synthesis
- elongates a new nucleotide strand 5’ to 3’ from the 3’ OH group provided by the primer
- large complex aka a holoenzyme
- synthesizes only in 5’ to 3’ direction so synthesis along an advancing replication fork occurs in one direction on one strand and in the opposite direction of the other
- READS DNA 3’ to 5’
- has 5’ to 3’ polymerase activity
- has 3’ to 5’ exonuclease activity (allows correction of errors)
Telomeres Function
- maintain ends of chromosome
1. Structural- serve as a cap on end of chromosomes to block unraveling
2. Replication of ends - generally does not occur in somatic cells (shortened to death)
- single celled organisms and germ cells do have to deal with this
What is the function of hydrogen bonds in DNA?
- holding the 2 strands together by linking the bases
Histone tails
- targets for binding in chromatin remodeling
How is DNA packed into the nucleus
- assembly of nucleosome when histone proteins attach to DNA in the molecule
- nucleosome is a loop of the DNA and proteins
Where are the nitrogenous bases attached in B form DNA
- to the 1’ carbon of the sugar
Capping consists of:
- addition of an extra nucleotide at the 5’ end
- addition of a methyl group to the base of the added nucleotide
- methylation of 2’ OH of sugar of nucleotides at 5’ end
why do you need dNTPs for DNA replication?
- they provide energy
RNA coding sequence in transcription
- the template DNA strand
- Actual sequence of nucleotides to be transcribed into RNA
Transcription requires
- ssDNA template
- Substrates to make RNA (ribonucleoside triphosphates)
- Transcription machinery
Nucleosome
- two copies of H2A, H2B, H3 and H4 + DNA (histone proteins and DNA)
- Bead and string model
- Beads = 8 histones (2 tetramers) (only 4 diff types)
- String = 2 strands DNA (200 bps total)
5’ end of DNA strand
- phosphate group
What does the DNA backbone consist of
- phosphate group and sugar
Lampbrush Chromosomes
- only seen in meiosis
- represents areas where we need to ACCESS DNA!!
- characterized by extended lateral loops
- extended uncoiled versions of meiotic chromosomes
- each chromosomal loop is composed of one double helix of DNA
- each central axis is made up of two DNA helices
- DNA that’s pulled out and unwound a little bit so you can access it
- after it repacks very tightly and continues through meiosis
- similar to polytene puffs
Licensing of DNA replication
- replication licensing factor attaches to the origin of replication
- ONLY AFTER THIS HAS HAPPENED can initiator proteins start functioning
- When initiator proteins start replication, they lose their licensing factors & cannot bind & start replication again until after replication is complete, after G1
How are proteins encoded in DNA? if every nucleotide encoded a different amino acid, if every two nucleotides, and if every three nucleotides
- Francis Crick
- if every nucleotide only 4 options AUGC
- if every two nucleotides 4x4=16 possibilities
- if every three nucleotides 4x4x4=64 possibilities
Tobacco Mosaic Virus (TMV) Demonstrated?
- when purified RNA from Tobacco Mosaic Virus was spread on tobacco leaves, the lesions caused by the viral infection appeared
- proved that RNA is also genetic material of viruses, rather than protein
1) Start with Type A and Type B tobacco mosaic viruses (both are strands of different RNA surrounded by different protein)
2) Remove protein coat so RNA is the only thing left
3) put some of Type A virus into Type B soln, and Type B virus into Type A soln
4) This makes Hybrids with Type A RNA surrounded by Type B protein, and Type B RNA surrounded by Type A protein
5) Later, RNA replicase was found in bacterial phage
DNA replication unwinding step
- proteins stabilize the unwound helix and assist in relaxing the coiling tension created ahead of the replication fork
- DNA helicase
- ssDNA proteins
- DNA gyrase
Splicesosome
- RNAs and Proteins
Stop codon
- UAA, UAG and UGA
- no tRNA can pair with these
Upstream vs. Downstream in transcription
Upstream = anything before the promotor sequence on a gene Downstream = anything after the promotor sequence on a gene
Two kinds of nitrogenous bases
- Purines
2. Pyrimidines
Avery, MacLeod and McCarty’s findings
- Found that the transforming principle is DNA
- furthered Griffiths findings
1) Mixed virulent S BACTERIA with RNase (kills RNA), Protease (kills proteins), and DNase (kills DNA) to see what was destroyed and what wasn’t
2) Only DNase destroyed the transforming substance, so it was found that the transforming substance is DNA - RNAse killed RNA but bacteria was still virulent
- Protease killed proteins but bacteria was still virulent
- DNAse killed DNA & bacteria was no longer virulent =genetic material
- their publication on the chemical nature of the “transforming principle” in bacteria was the initial event that led to the acceptance of DNA as the genetic material
Purines
- nine member DOUBLE ring
- adenine and guananine
negative supercoil
- removing rotations from circular DNA so that it takes less energy to open up the DNA for replication
Polytene Puffs
- the uncoiling events result in a puff
- visible manifestations of gene activity (transcription that produces RNA)
- Areas of the polytene chromosome that are opened up to be able to access DNA
and undergo gene transcription - this opening creates a “puff-like” structure
Eukaryotic Evidence that DNA is genetic material
- Nucleic acid and proteins are found in the nucleus
- DNA also found in mitochondria and chloroplast - amount of DNA correlated with number of chromosomes (ploidy), protein is not
- Nucleic acids absorb UV in same spectrum that causes mutation
Transcriptional Unit
- stretch of DNA that codes for an RNA molecule and the sequences necessary for its transcription Includes: -promoter -transcription start site -RNA coding region -termination site
Sigma factor
- added to core subunits
- holoenzyme which will initiate transcription at promoter
- different sigma factors direct initiation at different promoters
- scans the DNA and looks for the promotor sequence to begin transcription
- Proteins that function in prokaryotic transcription initiation.
- Allows RNA polymerase to bind to promoter region to initiate transcription.
- SPECIFIC to type of gene
Telomere Problems
- replicative enzymes can not replicate ends of chromosomes
2. chromosomes would get shorter to the point of big problems
Point centromere
- small centromere, less complex
TERRA (transcribed regions)
- telomeric repeat containing RNA
- contributes to methylation
- repeats can be very big
- Help shut down ends of chromosomes, making them heterochromatic, so you don’t lose DNA
Major and Minor grooves
- repeating and alternating structures that act as base pair specific (major) and unspecific (minor) recognition
- binding sites for proteins
When transcription happens:
RNA synthesized in its 5’ to 3’ direction…
THIS IS THE DIRECTIONALITY
- DNA template read in its 3’ to 5’ direction
In what form do the bases pair with each other in B DNA
- they base stack on top of eachother
RNA pentose Sugar
- ribose
- contains OH on 2’ carbon
snRNP
=RNA - small nuclear RNAs + proteins
- small ribonucleprotein particles
Consensus sequences in prokaryotes (define and 2 types)
- sequences of the DNA PROMOTOR that bind to the RNA polymerase holoenzyme
- the sequence of nucleotides in DNA or amino acids in proteins most often present of a particular gene or protein under study in a particular organism (little variation in different genes)
1. -10 consensus sequence= Pribnow Box - site where unwinding begins
2. -35 consensus sequences
Prokaryotic Replication Process
- Initiation - initiator proteins bind to oriC, relaxing strand, breaking H-bonds, and beginning replication
- Helicase and ssbinding proteins bind to the strand to unwind it
- Small replication bubble starts opening/unwinding double helix
- DNA gyrase works ahead of replication fork to reduce strain & prevent supercoiling
- Primase puts down RNA primers to provide a 3’ OH group for the attachment of DNA nucleotides
- DNA polymerase III elongates a new nuclotide strand 5’ to 3’
- DNA polymerase I replaces RNA primers with DNA
- Ligase joins the okazaki fragments by sealing nicks in sugar-phosphate backbone of new DNA
Z-DNA form
- left handed helix
- zigzag backbone
- sites of active genes can make Z-DNA
RNA editing
- alters nucleotide sequence of mRNA
Eukaryotic Transcription Initiation
- Promoters
- found within or adjacent to the gene it regulates - Enhancers
- affect transcription, can be thousand of base pairs from the gene
- Small pieces of DNA that increase the likelihood that transcription of a particular gene will occur
- bound to proteins called transcription factors
Retroviruses
- RNA molecules that use reverse transcriptase(RNA back to DNA) to integrate into the host genome
Chromatin remodeling
- necessary for replication machinery to access DNA
Wobble
- The first 2 ribonucleotides of triplets are more critical than the 3rd in attracting the correct tRNA during translation
- WOBBLE = a more flexible set of base-pairing rules at the 3rd position of the codon that do not necessarily alter the resulting amino acid
- EX:UUU and UUA both code for the amino acid phenylalanine
Q-banding
- a chromosome banding technique that uses UV light
- Makes the bands glow which helps you see if the chromosomes have the correct banding pattern
Synonymous codons
- Codons in the genetic code that all encode the same amino acid
start codons/Initiators for protein synthesis in BACTERIA
- AUG encodes for N-formyl-Methionine to start translation (RNA –> proteins)
- usually removed from protein
chromatin
- complex of DNA and proteins that make up uncoiled chromosomes
- in eukaryotic chromosomes interphase nucleus stage
Frederick Griffith
- Transforming principle
- experimented with different strains of Streptococcus pneumonia
- studied a virulent strain(disease causing) of pneumonia (S=smooth coat) and an avirulent strain(did not cause disease) of pneumonia (R=rough coat)
- S killed mice, R did not kill mice
- Heat-killed S did not kill mice, but Heat killed S + R DID kill mice (shouldn’t have bc each individual component didn’t kill on its own)
- concluded that some avirulent (R) could transform into virulent (S) to become virulent and kill mice
- due to TRANSFORMATION
DNA replication in eukaryotes Priming Step
- DNA polymerase
- Primase
Eukaryotic DNA replication vs Bacterial replication
- multiple replication events at the same time on the same chromosome
- large variety of DNA polymerases
- Nucleosome assembly makes more of a mess
- Linear not circular chromosomes
Messelson and Stahl Experiment
- grew E. coli in heavy nitrogen(N-15) and light nitrogen (N-14)
- DNA in N15 medium was heavily labeled with N15 and sunk to bottom of test tube
- DNA in N14 medium replicated & the new DNA was made with the light N14 and floated near the top of the test tube
- DNA in mixed media N14/N15 created hybrid strands of N14/N15
- showed that semiconservative replication occurs in PROKARYOTES
Transcription and nucleosomes eukaryotic transcription
- Histone Acetyl Transferases (HATs)
2. Methylation of Histones and DNA
DNA polymerase 1 in prokaryotes
- replaces RNA primers with DNA
- DNA polymerase 1 has 5’ to 3’ polymerase activity
- has 3’ to 5’ exonuclease activity (BACKWARDS) to proof read and correct errors
- has 5’ to 3’ exonuclease activity (forwards) to remove primers
What do the lampbrush loops tell us?
- represent DNA that has been reeled out from the central chromomere axis during transcription
Process of Transcription
- Only open up ONE strand, read the complement base from 3’ to 5’, and make RNA 5’ to 3’
- IGNORE the other strand
Heterochromatin
- highly condensed chromatin in interphase (silent)
- inactive chromatin in areas where there are few or no genes
Histones
- POSITIVELY charged basic proteins found in chromatin
