Exam Review Flashcards
Watson and Crick suggested that the double helical DNA model suggests a _________ copying mechanism which means…
Semi conservative
after one round of DNA replication, each DNA contains 1 parental and 1 newly syntheized strand
What are the 3 models of DNA copying mechanisms
- Conservation replication
- Dispersive Replication
- Semi-conservative Replication
What are the 3 modes of DNA replication
- Theta replication
- Rolling circle replication
- Linear chromosome replication
Describe theta replication
- Replication that occurs in mostly circular DNA
- double stranded DNA unwinds at the replication origin
- This produces single stranded templates DNA synthesis and replication bubble forms with a replication fork at each end
- the fork then proceeds around the circle bidirectionally
- Eventually two circular DNA molecules are produced
Rolling Circle Replication
- Specialized form of replication that occurs in the F factor and some viruses only
- replication is initiated by a break in one of the nucleotide strands
- synthesis begins at the 3’ end of the broken strand; the inner strand is then used as a template. the 5’ en d of the broken strand is displaced
- cleavage releases a single stranded linear DNA and a double-stranded circular DNA. Replication is unidirectional.
- the linear DNA may circularize and sere as a template for the synthesis of a complementary strand
linear replication
- Linear chromosome replication occurs in the linear chromosomes of eukaryotic cells.
- This DNA replication is bidirectional.
What are the requirements for DNA replication (4)
- DNA dependant DNA polymerase
- Four deoxyribonucleoside triphosphates (dNTPs)
- A single stranded template of DNA to be copied
- An RNA primer (provides the 3’ OH end to initiate DNA synthesis by DNA polymerase)
DNA is ALWAYS synthesized in what direction?
5’ -> 3’
DNA synthesis is ____ on the leading strand and ___ in the lagging strand
continuous, discontinuous
Fragments formed during synthesis of the lagging strand is called
Okazaki fragments
What are DnaA proteins involved in prokaryotic DNA replication
- They are initiator proteins that bind to oriC (the origin)
- This causes a short stretch of DNA to unwind
DNA Helicase (Prokaryotes)
- Unwinds the DNA in the 5’ to the 3’ direction
- Only travels on the lagging strand
Single Strand binding proteins (Prok.)
Coats unwound single stranded DNA to protect and keep DNA single-stranded (prevents hairpins)
DNA Gyrase (prok.)
- A topoisomerase that makes double stranded breaks in DNA to relive the torsional strain that builds up from the activity of the DNA helicase.
____ requires a primer to initiate synthesis ___ does not
DNA polymerization, RNA polymerase
Primase (RNA Polymerase) (prok.)
Synthesizes short RNA primer that provides the 3’ OH end for DNA polymerase to begin DNA synthesis
Primase binds to ___ to initiate synthesis
Helicase
DNA Polymerase (prok.)
Elongates the new polynucleotide strand by catalyzing DNA polymerisation.
T or F: DNA polymerase requires a primer
True
DNA Pol III (prok.)
- The main DNA Pol
- has 5’-> 3’ polymerase activity and 3’ to 5’ exonuclease activity.
- can add dNTP on the 3’ end and also back up to remove a nucleotide that has been mis incorporated
DNA Pol I (prok.)
- Similar to DNA Pol III, but has 5’ -> 3’ exonuclease activity so it can remove the RNA primer and replace it with DNA
DNA Ligase (prok.)
Makes phosphodiester bonds between 5’ phosphate and the 3’ OH froup and seals the nicks in the lagging strand
Helicase (Euk.)
- Binds to initiator protein on double streanded DNA
- involves the MCM complex of proteins
In eukaryotes DNA replication only occurs during ___ phase
S
Polymerase Alpha (euk)
- Has primase activity
- generates the RNA primer
Polymerase Epsilon (euk)
preforms leading strand replicaiton
Polymerase delta (euk)
Preforms lagging strand replicaition
when DNA is doubled in euk. histones must be ___ and the modifications on them need to be replicated
doubled
The telomere problem
- The chromosome end will be degraded causeing shortening every round of replication.
- leaves gaps in DNA that are not replicated causing shortening
Telomerase
- Activity extends eukaryotic chromosome ends in replicating cells
- can extent the 3’ end without use of complementary DNA template
- results in shortening telomeres over time
Shorter telomeres is associated with…
Cellular senescence and death
What makes RNA less stable than DNA?
The presence of 2’OH groups in ribose causes it to react intramolecularly with the 3’OH site resulting in phosphate breakage.
What is transcription
Synthesis of RNA from DNA
In what Direction is RNA synthesis
RNA is synthesized in the 5’ to 3’ direction using the 3’ to 5’ DNA template strand.
The precursors to RNA syntheis are..
Ribonucleoside triphosphates (rNTPs)
RNA Polymerase Holoenzyme Subunits
- alpha
- beta
- beta-prime
- omega
What is the function of the alpha subunit?
Involved in the assembly of the tetrametric core
what is the function of the beta subunit?
Contains the ribonucleoside triphosphate (rNTP) binding site
What is the function of the beta prime subunit?
Contains the DNA binding region
What is the function of the omega subunit?
It helps to stabilize the tetrameric core
What is the function of the sigma subunit
- it binds to the RNA polymerase tetrametric core and assists in the correct initiation of transcription specifically at the promotor region of the prokaryotic gene. Many types of sigma factors -> allow for specificity.
- recognizes and binds to the -35 and -10 consensus sequances in the promotor region, properly positioning the RNA polymerase to begin transcription
Why is the -10 consensus sequence prone to unwinding?
Due to its AT rich content
Describe initiation of transcription in prokaryotes
- holoenzyme binds to the promoter and RNA polymerase is positioned over the transcription start site and has unwound the DNA to produce a single stranded template
- the orientation and spacing of the consensus sequence on a DNA strand determine which strand will be the template for transcription and therby determine the direction of transcription
- RNA polymerase binds, unwinds and joins first 2 nucleotides
Does RNA synthesis require a primer
no
Elongation
- Occurs when sigma factor is released and RNA polymerase begins to move along the 3’ to 5’ DNA template strand.
- Complementary nucleotides are added
- Localized DNA unwinding ahead of RNA polymerase and generates a “transcription bubble”
- Transcription bubble moves along with the unwound DNA rewinds behind the bubble
RNA polymerase has both helix ___ and ___ activities
unwinding, rewinding
Termination
- stop when RNA polymerase reaches the terminator region of the gene
- occurs upstream of where the actual termination takes place
- the newly synthesized RNA together with the RNA polymerase are released
What are the two major types of terminators in bacterial cells?
Rho-dependant (requires the Rho factor) and Rho-independant (aka intrinsic terminators)
Describe Rho-dependant Termination
Sequence Features:
- DNA sequence of terminator site causes polymerase to pause
- DNA sequance upstream of terminator encodes a stretch of RNA that is C rich and devoid of secondary structure
a) called the rho utilization
b) rho binds to rut site
- Rho moves alonf RNA towards paused polymerase
- rho factor has helicase activity
- unwinds the RNA-DNA hybrid
- ends transcription
Describe Rho-independent termination
Common features
- contains inverted repeats
- a string of 6-9 A’s follows the inverted repeats
- poly A sequance is transcribed into a poly U tail after hairpin is transcribed
- causes polymerase pause
- hairpin forms and destabilizes the DNA-RNA hybrid
- assisted by weak A-U base pairing
true or false: Pol I, II, and III are inly recruited to their promoter specific accessory proteins
true
Initiation in Eukaryotes
Involves a step wise assembly of general Transcription Factors of Pol II
- Tata box is the fist to assemble then followed by the remaining general TFs and Pol II
- assembly of the TFs and Pol II causes 11-15 bp of surrounding DNA to unwind
- template strand positioned within active site of RNA pol
Is the pre-initiation complex sufficient to initiate transcription?
yes
what is a mediator
involves multi-subunit that permits interactions with other activator/repressor proteins bound to upstream/downstream regulatory regions or enhancer sequences
Elongation in Eukaryotes
- Polymerase moves along the template strand leaving transcription factor intact at the promoter for reinitiation of transcrption with new polymerase
- RNA pol maintains 8nt transcption bubble
- DNA-RNA hybrid bends at right angle
- positions the OH group at the active site where nucleotides are added to the 3’ end of the growing chain
- newly sythesized RNA is seperated from DNA and exits through another cleft
Termination in RNA Pol I
Requires a termination factor similar to the rho factor in prokaryotes
Termination in RNA Pol III
Ends after transcribing a terminator sequence that produces a string of Us that is downstream of hairpin. Similar to rho-independant termination
Termination in Pol II
Transcription continues past termination sequence
- RNA is cleaved at consensus sequence in the RNA
Termination in Eukaryotes
- Cleaved RNA results in 2 RNAs: one that will encode a protein and the other with its 5’ trailing out of the RNA polymerase
- Rat 1 (5’ -> 3’ exonuclease) attaches to the 5’ end and chews up the RNA
- rat 1 does not bind to the 5’ end of the preotein coding mRNA because it is protected by a 5’ cap
What is Colinearity
- the coding region in prokaryotes that is uninterupted
- The sequance of the gene directly corresponds to the amino acid sequance
the number of nucleotides is proportional to the number of amino acids (3:1)
The shine delgarno sequance is found in ___ (prok or euk)
Prokaryotes
What are the three primary regions of a mature mRNA
5’ Untranslated Region (5’UTR): Does not code for amino acids, binds the ribosomal complex
Protein Coding Region: Comprises the codons that specify the amino acids begins with a start codon and ends with a stop codon
3’ UTR: does not code for amino acids. Affects the stability of the mRNA and regulates its translation
Exons are…
Protein coding segments
Introns are…
inervating (non-coding) segments
removal of ___ along with additional RNA processing steps are required to form the mRNA that will be translation into ___
introns, polypeptide
What is required to remove the intron?
the splicosomal complex which contains small nuclear RNAs and proteins
Where does splicing take place?
In the nucleus
What are the 3 main processing steps in eukaryotic nuclear pre-mRNA
- the addition of 7-methyl guanosine cap. linked betwen the 5’ phosphate of the 7 MG and the 5’ phophphate of the first RNA
- addition of a poly A tail to the 3’ end by poly A polymerase
- Removal of introns from pre-mRNA called splicing
Describe the 5’ cap
Methyl groups are added to:
- 7’ position of the terminal guanine
- 2’ OH group of the sugar of the first and sometimes second nucleotide
- to adenine if it is the second nucleotide
Descibe the 3’ poly A tail
50-250 As added to the 3’ end
- A are added without a template (polyadenylation)
- Poly A consensus sequance (AAUAAA) is the polyadenylation signal located 11 to 30 nucleotides upstream of the cleavage site
- affects stability of mRNA, facilitates attachment of the ribosome and export out of the nucleous
What is the polyadenylation signal
AAUAAA
Descibe RNA splicing
- removal of introns from pre-RNA
- Removal of introns must be precise in order to properly fuse the 3’ end of one exon to the 5’ end of the next exon
How does the eukarotic cell ensure that introns are properly removed?
Every intron has 3 conserved sequances thata re required for its precise removal:
- 5’ splice sequances containing the junction seequances GU (5’ end of the intron)
- 3’ splice sequence containing the junction sequence AG
- Intron branch point: a conserved A residue is located 18-40 nt upstream of 3’ splice site
What are the Steps of RNA splicing
- Pre-mRNA is cut at the 5’ splice site and the 5’ end of this intron attaches to the branch point (Lariat)
- 5’ phosphate of the G nucleotide binds with the 2’ OH group of the A nucleotide at the branch point
- Cut is made at the 3’ splice site and simultaneously the 3’ end of exon 1 becomes covalently attached to the 5’ end of exon 2
- intron is released as a lariat and degraded
Describe a Spliceosome
- RNA/ pritein structure
- contains 5 small nuclear RNAs
- The snRNAs associated with about 300 small proteins to form small nuclear SNURPS
What is alternative splicing?
- an alternative processing pathway
- pre-mRNA is processed in different ways to produce alternative types of mRNA
- alternative exons are used
- results in different proteins from the same DNA sequance (unless in the UTR region)
Describe the concept of multiple 3’ cleavage sites?
- alternative procssing pathway
-Can generate longer or shorter exons
- may or may not produce a different protein. depends if upstream or downstream of stop codon
- note that this is not the same as the 3’ splice site
What is Calcitonin
an alternative processing pathway
regulates calcium concentration in the blood
what is CGRP
an alternative processing pathway
Causes dialation of blood vessels and the transmission of pain
How is RNA editing carried out?
- Carried out by guide RNAs that can be made in the cell
- gRNAs direct the insertion of urodine bases into the mRNA by a repair polymerase
- This perminatly modifies the mRNA by making new codons that specify new amino acids
What is the editing of Apoplipoproteins B mRNA
- Apoplipoproteins are blood proteins that carry lipids
- RNA editing by cytidine deaminase changes C to U converting a normal glutamine codon to a termination codon which truncates the protein and gives it a different function with respect to lipid binding
Describe tRNA modifications
- the anticodon of the tRNA base pairs with the codon of mRNA
- The amino acid is covalently attached to the 3’ end of the tRNA
- tRNAs often contain modified ribonucleotides through the action of tRNA modifying enzymes
What processing events do tRNAs undergo
- removal of extra 5’ and 3’ sequances
- removal of introns
- nucleotides 5’-CCA-3’ are added to the 3’ end of the tRNA for all tRNAs
- modification of several nucleotides
What is a ribosome
- comprised of 2 subunits and has a key role in protein synthesis including the formation of peptide bonds between amino acids