Lecture 15 - Nucleic Acid Synthesis Flashcards
What is replication?
Copying of parental DNA to form daughter DNA molecules with identical nucleotide sequences
What is transcription?
Parts of the genetic message encoded in DNA are copied precisely into RNA
What is translation?
The genetic message encoded in mRNA is translated on the ribosomes into a polypeptide with a particular sequence of amino acids
Does DNA have parallel or anti-parallel strands?
Anti-parallel
What is the function of chromatin?
To package DNA in a more compact shape
What is Phe-tRNA used in?
Protein synthesis
What is a hammerhead ribozyme?
An RNA enzyme
Describe the conservative theory of DNA replication
The parental molecule is conserved, or remains intact
Describe the semi-conservative theory of DNA replication
The parental strands separate but they are conserved as strands
Describe the dispersive theory of DNA replication
The parental strands are fragmented and interspersed with new DNA
Does the conservative, semi-conservative, or dispersive theory occur in reality?
Semi-conservative
Describe the Meselson-Stahl experiment
1) E. coli cells were grown in only heavy nitrogen (N15) in order to label all of the nitrogens in DNA w/ heavy nitrogen
2) Cells were isolated and then transferred to a medium with only light nitrogen (N14) and grown for one generation and then DNA was extracted
3) Continued to grow cells in light nitrogen and DNA was extracted after each generation
4) DNA was analyzed by CsCl density centrifugation - DNA with heavy nitrogen will accumulate further down the tube (where its density matches the CsCl density) and light nitrogen will be higher up
5) Each subsequent generation has a bit more light nitrogen
What is the key enzyme in DNA replication?
DNA polymerase
Do prokaryotes or eukaryotes have a more complex method of DNA replication?
Eukaryotes
What are 3 properties of all polymerases?
1) Use dNTP’s as substrates
2) Require a primer and a template
3) Elongate in the 5’ to 3’ direction
What happens to the primer during DNA synthesis?
Primer is elongated using the template to direct the nucleotide to be incorporated in a base pairing sense (G + C and A + T)
What happens when a dNTP bonds to a primer?
Pyrophosphate is lost b/c the nucleotide only needs one phosphate
Why must bases be properly paired?
If not, it can exclude them from the active site of DNA polymerase
What is the function of RNA polymerase in DNA replication?
RNA polymerase opens up the DNA to make a loop at a specific point and can initiate elongation without a primer
What is the major difference between RNA polymerase and DNA polymerase?
RNA polymerase can initiate elongation without a primer
What are okazaki fragments?
Small fragments of single stranded DNA that have nucleotides of RNA at the 5’ end, which serves as a primer for DNA polymerase
What happens to the leading strand during DNA synthesis?
It is elongated 5’ to 3’ continuously to the “end”
What happens to the lagging strand during DNA synthesis?
It is synthesized as a series of small fragments that start whenever ~1000 nucleotides of single-stranded DNA is opened up by growth of the leading strand
The RNA primer is removed by ____
RNase
How are okazaki fragments joined into one continuous strand?
By DNA ligase
Do okazaki fragments occur on one or both strands of DNA?
Can occur on both
What are the 2 experiments that proved that DNA contained all the genetic information?
1) Avery-McLeod-McCarty experiment
2) Hershey-Chase experiment
What 4 facts were proven in the Griffith experiment?
1) A mouse infected with the virulent bacterium died
2) A mouse infected with the mutant (no capsid) lived
3) A mouse infected with the heat killed batch also lived
4) A mouse infected with a mixture of the mutant strain and the heat killed batch died
What questions arose from the findings of the Griffith experiment?
- Was the non-virulent mutant resurrecting the heat killed bacteria?
- Was something in the heat killed bacterium transforming the non-virulent to virulent?
What did the Avery and McLeod experiment show (keeping in mind the results of the Griffith experiment)?
- Only the fraction with nucleic acids caused the non-virulent bacteria to have virulent properties
- DNA from the heat killed strain was recombined into the chromosome of the non-virulent strain, replacing a defective gene for capsid synthesis with a normal gene
What was the question posed by the Hershey-Chase experiment?
Whether the protein or the DNA entered the cell on infection, and passed on the genetic info of the phage
What did the Hershey-Chase experiment study?
A bacteriophage that infects E. coli
Is the RNA that is produced from transcription an opposite or a duplicate of the DNA strands?
It is the opposite of one, and a duplicate of the other (except all of the T’s become U’s)
What are 3 requirements of RNA polymerase for transcription?
1) NTP’s
2) Template (DNA)
3) Promoter and terminator
_____ signals where the RNA polymerase is to start and stop
The DNA sequence
What must happen after synthesis of the primary transcript for mature mRNA to be produced?
Processing
What are the 2 main forms of RNA polymerase that exist in bacteria?
1) Core enzyme - carries out elongation of the RNA chain after sigma release
2) Holoenzyme - binds to the promoter site on DNA to initiate transcription
Is a primer needed for transcription?
No, the holoenzyme recognizes and binds to the promoter sequence on the DNA
What is the key protein factor of transcription and what is it required for?
- Sigma
- Required for binding to the DNA at the promoter
What must happen to the sigma protein for elongation to begin?
It must leave the complex
Which sequences are recognized by the holoenzyme?
-10 and -35
Which sequence facilitates the formation of the open complex and why?
-10 sequence because it is AT rich
Does RNA polymerase travel at the same speed across the DNA sequence?
No, it speeds up and slows down depending on the DNA sequence
What happens if the RNA polymerase pauses for too long?
Termination occurs
What causes termination of transcription?
The DNA sequence, because it causes the enzyme to slow, and then other factors facilitate termination
What are the 3 other factors (besides DNA sequence) that facilitate transcription termination?
1) Loop structure
2) Rho protein
3) Poly U
What causes a loop structure?
Palindromic (same forwards and backwards) sequence in the new RNA
What does a loop structure cause?
The RNA-DNA duplex being disrupted, weakening the complex and making dissociation of the RNA from DNA easier
What is rho protein?
A termination factor that binds to the 5’ end of the RNA transcript and “chases” after the RNA polymerase, catching up to it when the RNA polyermase is paused and then destabilizes the RNA-DNA duplex causing dissociation of RNA from DNA
What is poly U?
A sequence of 5-10 uridines in a row at the end of the RNA, destabilizing the RNA-DNA duplex making dissociation easier
What are the 3 types of RNA polymerase that are needed for eukaryotic transcription and what does each do?
1) RNA polymerase 1 - makes rRNA
2) RNA polymerase 2 - makes mRNA for translation
3) RNA polymerase 3 - makes tRNA, some rRNA and other specialized RNA
Does transcription or translation take up more energy?
Translation
