Gene Expression and Replication Flashcards
Semiconservative model of DNA replication
Each strand of template DNA used to synthesize new strand + each template remains annealed w/new strand
DNA polymerases
Enzymes that synthesize new complementary DNA strands - most can only extend pre-existing nucleic acid strand at 3’ end of molecule by adding nucleotides
Replication bubbles
Suggest presence of replication forks on each side of bubble where enzymes operate to replicate new DNA
Okazaki fragments
Small new single-stranded DNA molecules that suggest that DNA synthesis at each replication fork is continuous on the leading strand while it’s discontinuous on the lagging strand
What did Okazaki conclude?
That DNA replication is semicontinuous
RNA primers
Fragments at 5’ end consisting of RNA
Primase
Synthesizes RNA primers
DNA replication in 3 quick steps
- Initiation
- Elongation
- Termination
Enzymes involved in DNA replication
- Helicase
- Single strand binding protein
- Primase
- DNA polymerase
- Topoisomerase
- Exonuclease
- Tus protein
Helicase
Separates + unwinds DNA strands
Single strand binding protein
Prevents separated parental strands from re-annealing after being separated into 2 single strands by helicase
Topoisomerase
Cleaves 1/both of parental DNA strands to relieve tension caused by supercoiling of strands due to movement of replication forks - changes DNA topography (degree of unwinding)
Exonuclease
- Removes RNA primers from Okazaki fragments - when necessary it removes improperly incorporated nucleotide before resuming DNA synthesis (proofreading)
- Also removes RNA from lagging strand + replaces w/DNA as elongation continues
Tus protein
In prokaryotes only + binds to terminator site - prevents DNA unwinding by helicase which arrests movement of replication fork as it reaches Ter site
Terminator site
Sequence on bacterial chromosome that leads to arrest of helices + ends DNA replication
Replication origin/ori sequence
- Specific sequence at prokaryotic/eukaryotic DNA - generally bound by proteins + when cell division occurs additional proteins are recruited to ori site
- DNA replication starts once essential proteins have been recruited to the ori site
Prepriming complex
oriC + DnaA + DnaB + DnaC protein complex - once formed, DnaC released
Stringent control
Plasmids that divide only once per cell generation are under this
Relaxed control
Plasmids that replicate independently from cell division are under this - plasmids used in biotech are under this which allows for presence of several plasmids/engineered cell
2 steps of formation of protein complex which initiates DNA replication
- Formation of pre-replicative complex
- Activation of pre-replicative complex (occurs during S phase) - activated pre-replicative complex known as initiation complex
Pre-replicative complex
Forms during G1 phase of cell cycle
Origin recognition complex
In eukaryotes the ori site is 1st recognized by this group of proteins which recruits other proteins including MCM complex (acts as helicase) - once all protein in pre-replicative complex have bound ori site the pre-replicative complex is “licensed”
Kinases
- Required to phosphorylate several proteins during activation of pre-replicative complex - they are enzymes that phosphorylate other proteins
- Phosphorylation events in activation of pre-replicative complex leads to binding of primase, polymerase + replication protein A (single stranded binding protein in eukaryotes)
Why do primers contain RNA instead of DNA?
Higher error rate occurs during primer synthesis in comparison to error rate during elongation process - so RNA use allows for subsequent recognition + removal of primers from newly synthesized DNA strands + reduces error rate compared to expected error rate if DNA primers used
How does DNA polymerase catalyze the extension of the new DNA strand once loaded onto DNA?
It catalyzes the addition of nucleotides at 3’ end of new strand
How can DNA polymerase catalyze 4 dif specific reactions?
It recognizes substrates that form acceptable Watson-Crick base pairs w/template DNA strand bound to enzyme so it allows for rapid entry + exit of 4 possible dNTP molecules
Sliding clamp mechanism
- DNA polymerase is a clamp that remains open until dNTP enters + forms Watson-Crick base paired structure w/parental strand - clamp then closes thus catalyzing addition of this nucleotide to growing DNA strand
- Clamp slides to next position along parental DNA+ opens and is ready to catalyze next rxn
Nicks
Missing phosphodiester bonds between Okazaki fragments
Replisome
Proteins involved in DNA replication form this complex - it moves continuously w/out detaching from leading strand but periodically detaches from lagging strand to allow discontinuous synthesis of Okazaki fragments on lagging strand
Telomeres
Nucleotides at 5’ extremity of lagging strand which can’t be replicated - shorten as a function of # of cell doublings in adult animal cells
Telomerases
Can extend length of telomeres to counteract shortening that occurs upon each replication cycle
Fts proteins
Forms ring in centre of elongating cell that aids in chromosome segregation between 2 daughter cells
Mitosis
Occurs during M phase - asexual cell division in eukaryotes happens via this process + includes prophase (DNA condensation + mitotic spindle formation) + metaphase (chromosomes align) + anaphase + telophase (mitotic spindle disassembles nuclear membrane closes around segregated chromosomes + DNA condensation decreases)
Excision repair
Nucleotides (nucleotide excision repair) or nitrogenous bases (base excision repair) are removed + replaced by Watson-Crick base paired nucleotides
How does DNA glycosylase alter the nitrogenous bases of nucleotides?
It removes glycosidic bond between nitrogenous base + deoxyribose on DNA backbone - endonuclease then cleaves 1 side of deoxyribose residue + adjacent nucleotides removed by exonuclease (gap is filled by polymerase + nicks sealed by ligase)
RNA polymerase
Carries out RNA synthesis
Coding strand
Primary RNA transcript has same sequence as DNA strand complementary to this strand - sequence provided in 5’ to 3’ direction
Post-transcriptional modifications
Series of modifications that primary RNA transcript undergoes prior to obtaining mature mRNA - increase diversity of mature RNA molecules obtained from single primary transcript
Initiation in transcription
RNA polymerase binds to promoter sequences on DNA - RNA polymerase recruited to promoter indirectly via proteins that recognize specific promoter sequences
Promoters
DNA sequences containing sequence bound by RNA polymerase - define location of transcription initiation site + control rate of gene transcription
What are the proteins that recognize promoters?
Sigma factors (subunits of RNA polymerase that detach from other subunits when transcription initiated) in prokaryotes + transcription factors in eukaryotes
Elongation in transcription
RNA polymerase synthesizes RNA starting at initiation site - RNA elongation occurs via addition of ribonucleotides at free 3’ OH group of growing RNA strand
Termination in transcription
RNA polymerase removed from DNA + RNA synthesis stops - occurs due to presence of termination sequences on RNA chain
Holoenzyme
RNA polymerase is this in a prokaryote - it’s an enzyme composed of several protein subunits
Conserved sequences
Have high similarity between species
Consensus sequences
Have high similarity between genes
Pribnow box
Consensus sequence that’s highly conserved between species in prokaryotic cells
Upstream recognition sequence
Consensus sequence which determines type of sigma factor that associates w/the promoter
Efficient promoters
Lead to higher transcription rates
What happens once the RNA polymerase holoenzyme has bound the promoter?
- Promoter melts - 2 strands of promoter separate + RNA polymerase core protein binds to template strand
- Also sigma factor dissociates from RNA polymerase core enzyme
What is the first base present on template strand/5’ end of RNA chain?
- T residue / A residue
Regulatory sequences
Regulate transcription factor binding to promoter - eukaryotic promoters may include these upstream or downstream from transcription initiation site
What 3 types of RNA polymerase do eukaryotes possess?
- RNA polymerase I which synthesizes most rRNAs
- RNA polymerase II which synthesizes primary transcripts of mRNA
- RNA polymerase III which synthesizes tRNAs + 5s subunit of rRNA + other short RNA molecules
Housekeeping genes
Expressed constitutively + contain sequence rich in GC (GC box)