Define a DNA polymerase reaction and its components.
template, enzymes, dNTP, Mg++
Where does DNA replication begin? Where does it terminate?
- Ori C
- TUS in pro
- end of the strand in euk
- AZTTP for retro
Know about leading and lagging strand synthesis.
- leading: continuous synthesis, fast, poly III, one primer
- lagging: discontinuous, slow, poly III, poly I, DNA-ligase, multiple primers
How many primers are required for each strand synthesis?
1 vs 100,000
Primer (DNA or RNA) with 3’OH, a minimum of __ bases hydrogen bonded to template
What are Okazaki fragments? Remember some of the components required in lagging strand synthesis such as Pol I to _______ and DNA ligase to _______.
- short segment of DNA made on lagging strand
- remove RNA primers and add dNTP one by one
- join the Okazaki fragments
Know the polarity of DNA to be replicated.
- copying starts w/ 3'-end
- extend 5'->3'
Know the major eukaryotic DNA polymerases.
- I: slow, one-by-one, small, exnuclease
- III: large, fast, complex
- mtDNA pol
Remember clamp protein (____ in eukaryotes and ____ in prokaryotes) that _________.
- β clamp
- clamps DNA pol (mainly delta and epsilon) to template and speeds up polymerase rate
What does enzyme telomerase do? It has RNA component with sequence complimentary to telomeric( DNA) sequences.
- fix the 5' end problem
- use RNA template to extend the original template much beyond
- then use the extended DNA as the template to resume the duplication to guarantee the original parental info is copied.
_____ that convert supercoiled DNA to relaxed form
______ that separate two parental strands of DNA
Accessory proteins promoting tight binding of polymerase to DNA and thereby _______ (sliding clamps)
increase the speed of polymerases
Extension of RNA primer
DNA polymerase III
Removal of RNA primer and replacing w/ dNTP
DNA polymerase I, one nucleotide at a time
sealing of the nick for lagging strand
Open the DNA double helix
single-stranded DNA-binding protein
SSB, stabilizing the separated single-strand parental DNA before their daughter copies are produced
- origin of replication (also called the replication origin) is a
- particular sequence in a genome at which replication is initiated
[duplication initiation factor, binds to] Ori C
begins unwinding (helicase)
Gyrase and Topoisomerase:
Supercoil to relax transition
Synthesis of primer (RNA)
Termination of cyclic dsDNA synthesis
- termination signal: TUS factor
- Two forks reach each other at mid point of the circle and are stalled by TUS factor
- Replication completes with generation of catenated circles, which are separated by Topo IV
PROOF READING ACTIVITY OF POL I is acting as a
3’ to 5’ exonuclease
E.coli pol I structure
- 3’ – 5’ exonuclease
- G1: starts with one copy
- S phase - synthesis, duplicating
- G2: two copies made
- M: mitosis
unique Problem with Eukaryotic DNA replication and solution
- 5’ – RNA primer removal and fill up
Define RNA polymerase reaction. How does it differ from DNA pol reaction?
- no primer
- different polymerases
What are pro and eukaryotic promoter sequences? How are these recognized?(e.g. sigma factor)
- tataat pribnow box
- by sigma factor of RNA polymerase
- tata, gcgc, caat
- by transcriptional factors
What is the termination signal for RNA synthesis in prokaryotes?
poly-U, hair pin, rho-factor - C-rich region
How many eukaryotic RNA polymerases exist and what do they synthesize?
- I: rRNA
- II: mRNA
- III: tRNA
What is the one major difference in prokaryotic and eukaryotic transcription? How is primary transcript processed in eukaryotes?
- Eukaryotes: mRNA sites in nucleus, then it comes out
- Prokaryotes: process where it is made and then used and binds to ribosomes
- Primary transcript: eukaryotes do this, prokaryotes don’t
- polycistronic vs monocistronic
Remember capping at 5’end and poly A addition at 3’ends. Also know the processing of intron-exon sequences.
Which small RNAs do you recognize? Micro RNAs, Si RNAs, SnRNP, SL7 RNA
- miRNA: stem loop precursor, suppress
- siRNA: RNaseIII, silencing
- 7SL RNA: transport secretory protein
- snRNP: mRNA processing
- oligoA: interferon
Recognize rifamycin and alpha-amanitin targets.
- RNA polymerase (prokaryotic)
- RNA poly II (eukaryotic)
Prokaryotic RNA polymerase
- holoenzyme: a, a, b, b', w, s, s is used to locate the start point
- core enzyme: a, a, b, b', w, elongation of RNA chain
Major RNA species
- rRNA: Makes ribosomes
- tRNA: carry amino acids
- rRNA and tRNA account for 95% of all RNA
- mRNA: short, lots more of them
- small RNAs, U1-U6: uridine rich sequences; Participate in the splicing of RNA reactions in eukaryotes
- miRNAs: 15-20 nt very small, missed detection
Eukaryotic promoter-binding transcription factors: ______.
CTF, SP1, and TFIID
posttranscriptional modification of eukaryotic mRNA
- 5'-5' linkage of 7-methylG-cap
- 3' polyA tail
- splicing of the introns (snRNP needed, splicesome formed)
EUKARYOTIC RNA POLYMERASES
- RNA polymerase I : rRNA; in Nucleoli
- RNA polymerase II: mRNA; Nucleoplasmic
- RNA polymerase III: tRNA, 5S rRNA; Nucleoplasmic
- -110 -60 -25
- CAAT GCGC TATA (box)
prokaryotic mRNA can be
- typically 21 to 25 base long and is produced from stem loop structured precursor RNAs.
- Involved in destruction of specific gene transcripts and suppression of translation of specific mRNAs.
siRNA or RNAi:
- small interfering RNAs similar to miRNAS, produced by RNase III enzyme
- Functions in Silencing of mRNA
- RNA required in transport (to Golgi) of secretory
(small Nuclear RNAs) form RNPs required in processing of mRNA.
(20-50 nucleotide long) stretch of A nucleotides linked from 2’ to 5’. Aids in interferon action.
TRANSFER & RIBOSOMAL RNA SYNTHESIS
- Synthesized as long transcripts
- 16 S tRNA 23S 5S
- Cleaves at spacer sequences with specific RNase
- CCA ends are added to the 3’-ends of all tRNAs
- Modifications of some bases, e. g. methylation in both tRNA and rRNA
INHIBITORS/DRUGS OF RNA SYNTHESIS
1. Enzyme specific
2. DNA intercalating agents
3. Substrate analogs
- Rifamycin for E. coli enzyme
- a-amanitin for pol II
Down-regulation of the supply of initiator Met-tRNAi via ______
- causes Premature release of nascent
- polypeptide by imitating tyrosinyl-tRNA and targeting 50S, 60S
target 30S and Inhibits initiation
target 30S and Inhibits binding of AA-tRNA to A-site
Inhibits peptidyl transferase via targeting 50S
Inhibit translocation via targeting 50S
RICIN (castor beans)
targets 60S and Inhibits binding of AA-tRNA to A-site
inactivates eEF2 for ribosome translocation
Formation of aminoacytyl-tRNA
- AA+ATP+aminoacytyl-tRNA synthetase->enzyme(aminoacytyl-AMP)+PPi->2Pi
Hydroxylation: (Proline) in collagen, Endoplasmic Reticulum
Glycosylation: O-linked as with Ser/Threo- OH, in Golgi; N-Linked as in lysine, in ER