2.1 Expression of Genetic Information Flashcards
Alkaptonuria
- Disease characterized by dark urine
- Trait is inherited (genetic basis)
- Affected individuals deficient for enzyme responsible for oxidizing homogentisic acid
- Invented term “Inborn Error of Metabolism)
Beadle and Tatum Experiment
- Irradiated Neurspora spores to induce mutations
- Grew irradiated Neurospora on complete medium
- Grew individual spores on minimal medium to identify mutants
- Mutant samples tested on minimal medium supplemented with specific vitamins
- sample of mutant cell grew on medium suplemented with vitamins but not grow on one supplemented with a.a
- therefore there must be a deficiency on an enzyme leading to the formation of vitamins
- deficiency is in gene associate with pantothenic acid which is with coenzyme A
Vernon Ingram (1956) studied sickle cell anemia protein defects
- Used proteolytic enzyme (trypsin) to cleave normal and sickle cell hemoglobin polypeptides
- Identified fragments with two dimensional paper chromatography
- One fragment migrated differently
- Sequenced fragment and identified mutation
- Glu (-ve charge) to Val (neutral) amino acid substitution
- Results in sticky patches on molecule
- Form crystals that distort RBC
Pro of the use of mRNA
- separates storage of genetic information from information utilization
- Also permits amplification of gene information
- Proteins translated from mRNA on ribosomes
- 1 DNA can make many mRNAs (amplification)
3 Types of RNAs
- mRNA-transcribed from DNA template-codes for protein
- rRNA-structural and catalytic RNA in ribosomes
- tRNA-carry amino acids to ribosome and mRNA transcript
- tRNA and rRNA form secondary structures
Basics of Transcription
- RNA transcribed by DNA dependent RNA polymerase
- Polymerase binds to promoter with aid of transcription factors
- DNA temporarily unwound as polymerase moves along
- RNA transcribed in 5’ to 3’ direction
- 3’ OH group of nucleotide at end of growing strand attacks 5’ alpha phosphate of incoming nucleotide triphosphate
Experimental System for Studying RNA Transcription
Fluorescent bead at downstream end
Can measure force produced by enzyme
RNA Polymerase Facts
- High processivity: stays associated with DNA template with high efficiency
- Force produced by RNA pol is twice that of myosin molecule
- Energy derived from hydrolysis of NPPP (ribonucleotide) precursors
- Movement not continuous
- Polymerase may “stall”
- High Fidelity
- RNA “bactracks” to correct errors
Initiation of Transcription in Prokaryotes
- five subunits form a core enzyme
- purified core enzyme will attach anywhere. If you add a sigma factor though, it will attach to promoter sites
- Sigma (σ) factor increases affinity for promoter sites
- complete enzyme = holoenzyme
- once it susessfully binds and starts doing about 10-12 nucleotides it gets into a rhythm and becomes a trascipional elongation complex and loses the sigma factor
Bacterial Promoters
- Located in region preceding transcription initiation
- 35 bases upstream-TTGACA consensus sequence
- Recognized by σ factor
- 10 bases upstream-TATAAT
- “Pribnow Box”
- Identifies precise start for transcription
Eukaryotic RNA Polymerases
RNA Pol I - synthesizes large rRNA transcripts (28S, 18S, 5.8S)
RNA Pol II - synthesizes mRNA and small nuclear RNA (snoRNA, snRNA)
RNA Pol III - synthesizes tRNA and small rRNA (5S rRNA)
RNA Pol IV - synthesizes siRNAs
(Plants only)
What are the components of a nucleosome
- gc (granular component)-Ribosomal subunits in process of assembly
- fc (fibrillar component) – DNA (genes) coding for rRNA
- dfc (dense fibrillar component) – rRNA nascent transcripts
Ribosomal RNA Synthesis
-christmas trees
-Called rDNA
-trasciption is evident by the fibrils attached to the DNA
no trascribable spacer is the area devoid of nascent RNA chain between adjacent molecules
-Short = closer to the site of transciption, the longer fibres are closer to being done
-the length between the shortest and longest is the single trascipton unit
-RNA and proteins work together to turn precursor rRNA into its final product and assemble it into subunits
-Eukaryotic ribosomes have 4 ribosomal RNAs 3 large 1 small. Large = 28s 5.8S 5S small is 18S
-Pre-rRNA carves 28s 5.8s and 18s. 5s is synthesized by a separate one
Kinetic Analysis of rRNA Synthesis and Processing
Nucleolar rRNA
45S converted to 32S
32S precursor to 28S and 5.8S
Cytoplasmic rRNA
18S rRNA appears before 28S rRNA
rRNAs are havily methylated so can use methyl donor thats radiolabled to see the process
45S is cleve and trimmed down to does 28s and 5.8S
other major product of 45S leaves the nucleus quite rapidly and appears in the cytoplasm as a mature 18s rRNA
Modification of Mammalian rRNA
- Large number of methylated nucleotides and pseudouridine residues in processed rRNA
- Posttranscriptional modification
- Conserved regions
- Regions that are modifired have conserved sequences that are recognized by snoRNA
