Genetic code and mutations Flashcards
What did Frederick Griffith do?
1930’s scientist that deduced genotypes and phenotypes can be transformed by injecting live wildtype cells and dead pathogenic cells into a mouse, discovering that the bacteria were capable of transformation.
What did Avery, Macleod and McCarty do?
determined that the material that caused the Tranformation in Griffith’s experiment was DNA as when they destroyed different macromolecules, all S strains survived except for when DNA was destroyed
What did Martha Chase and Alfred Hershey do?
Provided additional evidence that DNA was the mode of genetic material due to Radioactivity recovered in bacteria when injecting DNA with radioactive elemets.
What did the X-ray diffraction of DNA reveal about the structure of DNA?
Suggested DNA was long and skinny, with two similar parts running parallel to each other.
What is semiconservative replication?
Replication generates two identical duplexes (double stranded helices) of DNA where the two complemenary strands of the OG DNA separate and acts as templates for the synthesis of new strands.
What is the replisome?
DNA molecule and all its associated enzymes/proteins required for replication.
What are the steps of initiation of replication in bacteria?
- dnaA binds to high affinity dna A binding sites, which is used as an anchor to cooperatively bind ATP-dnaA complexes to the low affinity AT-rich region
- Topological stress breaks H-bonds and unwinds AT-rich region with the helps of HU, which provides a higher affinity to ATP-dnaA molecules
- ssb proteins stabilize single stranded DNA which prevents helix formation
- 6 monomers of dnaC recruits 6 monomers of dnaB onto each single strand
- dnaB monomers form a hexameric ring around each strand and dnaC monomers dissociate
- After helicase moves outward a bit, primase is loaded behind each helicase
- Sliding clamp is loaded between helicase and DNA polymerase III, while ATP-dnaA hydrolyzes to ADP-dnaA which promotes dissociation
- Primase lays down RNA primers that has 3’ OH ends that will be recognized by DNA polymerase III for DNA synthesis
What is the meselson and stahl experiment?
Used isotopes of nitrogen to rule out dispersive and conservative replication.
How does helixase bind in eukaryotes?
- Origin recognition protein binds to 11bp consensus sequence
- Cdc6 (helicase loader) attaches to Origin recognition protein
- First helicase and Cdt1 attach to origin recognition protein, then the second.
- Helix unwinds at AT rich region and Cdt1, Cdc6, and oirigin recognition protein dissociate
- As helicase begins to unwind at each side of the fork, a DNA polymerase is recruited to each
What are telomeres?
Non protein encoding repeated base pairs that elongate the shortened ends of synthesized DNA from lack of primer.
What are telomerases?
enzyme containing RNa that adds repetitive DNA sequences to the ends of telomere. It creates a T-loop and joins with shelterin protein complex, protecting the telomere from degradation
What is the hayflick limit?
Number of replication cycles in the cell’s life span (50-70) due to limit to the length of the telomere
What does telomere shortening cause?
Genomic instability, resulting in increased mutations, leading to cancers, dyskeratosis congenita (bone marrow faliure) and idiopathic pulmonary fibrosis (progressive lung disease)
What is telomere shortening caused by?
smoking, stress, poor overall health, chemical exposures, radiation
Who were elliot volkin and lawrence astrachan?
Observed RNA synthesis bursts in E/ coli following phage infection, followed by rapid decay of RNA. Suggested RNA may have a role in synthesizing more phages
What are types of non coding RNA and functions?
rRNA: translation, part of ribosomes
tRNA: translation, carries AA
snRNA: mRNA processing and intron removal
miRNA/siRNA: regulatory RNA
What is RNA polymerase?
Pairs template strand nucleotides with RNA specific complementary nucleotides (5’-3’)
How does RNA polymerase bind to the template strand of DNA?
Binds to promoter at -35 consensus sequence with help of sigma factor, then to pribnow box (-10)
What are the parts of RNA polymerase?
- 2 alpha units: assemble enzyme
- beta: catalysis
- beta prime: binds the DNA
- omega: assembles holoenzyme
- sigma factor: binds to promotor until transcription begins and initiates first unwinding
What is factor independent termination?
3’UTR region is transcribed with GC stretch, A rich stretch, and then GC stretch again. GC on either side form a stem-loop followed by a stretch of Uracils. Stem loop and poly-U destabilize RNA polymerase, inducing dissociation
What is Rho-dependent termination?
Rho factor protein recognizes and binds to rut site on on mRNA, moves along mRNA until it bumps into RNA polymerase, unwinding the last bit of DNA-RNA complementary base pairing and dissociating RNA polymerase.
How does RNA polymerase bind to DNA in eukaryotic transcription?
- TFIID (TATA-binding protein + TBP associated factor) binds to TATA box and forms initial committed complex
- TFIIA, TFIIB and TFIIF and RNA polymerase join initial committed complex
- TFIIE and TFIIH join the complex, forming the preinitiaton complex (PIC)
- When PIC is formed, transcription starts at the +1 nucleotide on the template strand.
What is the carboxyterminal domain?
Domain of tyrosine-serine-proline-threonine-serine-proline-serine sequences (YSPTSPS) in pre-mRNA transcript. Phosphorylation of serine in position 5 by TFIIH serves as signal for binding of 5’ cap.
How does RNA pol I, II and III terminate?
- RNA pol I: proteins bound to terminator elements on DNA
- RNA pol III: poly U stretch (factor independent termination)
- RNA pol II: torpedo model and allosteric model
What is the torpedo model?
Mechanism of transcription termination used by RNA pol II interacting with CTD serine 2:
- Transcription continues past cleavage site after pre-mRNA is cleaved
- 5’ end of new transcript is digested by Xrn2 until it chases down the polymerase, causing it to dissociate.
What is the allosteric model?
Mechanism of termination used by RNA polymerase II:
- transcription past cleavage site causes elongation factors to dissociate
- dissociation causes a conformational change in RNA polymerase II
- conformational change causes RNA polymerase II to dissociate from DNA
How is the 5’ cap added?
- RNA triphosphatase removes gamma phosphate from first pre-mRNA nucleotide
- Guanylyl transferase adds guanine to 5; end of the pre-mRNA
- produces 5’ to 5’ bond which forms a triphosphate linkage
- methylation of guanine by 7-methyltransferase finalizes cap.
What are the factors involved in polyadenylation of 3’ pre-mRNA?
- cleavage and polyadenylation specificity factor (CPSF)
- Cleavage stimulating factor (CStf)
- CFI, CFII
- Polyadenylate polymerase (PAP)
How is pre-mRNA polyadenylated?
- CPSF binds to cleavage site (AAUAAA) upstream of poly A site
- CStF binds to uracil rich downstream sequence element (DSE)
- CFI, CFII, and PAP join the complex
- Complex (CPSF) cleaves 15-20 nucleotides downstream of AAUAAA
- PAP polyadenylates from AAUAAA
