From Gene to Protein
Transcription
The Genetic Code
Translation
The Complex Proteome
Applied Lecture
Applied Lecture
Reading the Blueprint
DNA Molecule
Contains genes that code for proteins or RNA molecules, that have various cell functions
Information is stored in DNA of a cell
Stored information is interpreted and transmitted for cellular processes
Genes
Sections of DNA molecules, that contain formation that is transcribed into RNA copy
Either stays as RNa, or is further translated into mRNA, and makes up amino acid sequence, to make up proteins
Central Dogma
Process of copying and interpreting genes into proteins
Information that is stored in DNA, specifies sequence of amino acids in protein
DNA copied multiple times into mRNA
mRNA translated from nucleotide to protein
Gene Expression
Trasncription of information encoded in DNA to RNA
Transcription in Prokaryotes
DNA Transcribed from Template Strand
Nucleotide sequence contained in gene, will determine sequence of nucleotides contained in RNA transcript
RNA strand is same as template strand, due to base-pairing rule; except U replaces T
DNA Transcription Start & End Site
RNA polymerase attaches to DNA promoter regions
Promoter regions show transcription starting point
Transcribes in 3'-5' direction
Synthesized from 5'-3' direction
Terminator stops transcription
Transcription Initiation
Nucleotide sequence TATAAT is promoter sequence
Consensus sequence
Nucleotide sequence in DNA found at site
Also have TTGCCA
Enhance transcription rate
Transcription requires RNA polymerase and sigma factor proteins
Bind promoter region of DNA
Pair binds to create holoenzyme
Binds to and unwinds double-stranded DNA helix
RNA Polymerase
Elongates the forming RNA transcript
Separates DNA double helix
Ribonucleotides enter, assemble on DNA template strand
Passes template DNA through channel, transcribe template strand into complementary RNA
Restores DNA back into double helix
RNA Transcription Elongates at 3' End
Phosphate bond energy of incoming ribonucleoside triphosphate
Drive high energy reaction process
Required to create phosphodiester bond between incoming nucleotide and growing RNA transcript
Release and cleavage of pyrophosphate (phosphate-phosphate) group during phosphodiester bond formation, renders this polymerization reaction
RNA transcript elongation irreversible
Transcription Stopping
Termination sequence at 3' end
Requires nucleotide terminator
Releases RNA sequence
Releases transcription complex
Termination of Transcription
Rho-independent terminator
Inverted nucleotide repeat sequence
Fold back on themselves to form G-C hairpin loop along mRNA strand
Rho-dependent terminator
Rho factor - protein
Bind to and use ATP energy to move along RNA transcript while unwinding it from DNA template
Destabalize interaction between DNA and RNA template
Release of transcript & transcription complex
Transcription and mRNA Processing in Eukaryotes
Transcription in Eukaryotes
General Transcription Factors
Mediate binding of RNA polymerase to promoter
Initiate transcription
RNA Polymerase I
Transcribes genes for rRNA
RNA Polymerase III
Transcribes genes for tRNA
RNA Polymerase II
Transcribes mRNA
Template for production of protein molecules
RNA polymerase produces complementary, antiparallel strand of RNA
5' Cap
Post-transcriptional modifications
Add 5' cap and 3' poly (A) tail
5' end gets attachment of guanosine to mRNA through 5'-5' triphosphate linkage
7-methylguanosine
Adenine nucleotides added to 3' end is poly(A) tail
Follows polyadenylation signal sequence (AATAAA)
Termination
Poly(A)-dependent mechanisms
3' end modified by polyadenylation
Termination
similar to Rho-dependent factor
Termination
Similar to Rho-independent factors
terminated after termination signal
Processing RNA
RNA Splicing and Spliceosome
Amino acids that make up proteins, but do not code for anything
Introns
Exons
Sequence of amino acids in protein
Intervening sequence
Removed
Joined
spliced
Short nucleotide at each end of intron
Catalyzed by spliceosomes
Composed of 5 nuclear ribonucleoproteins (snRNP)
Recognizes complimentary base pairing
Finds splice site
Catalyzes reaction with hydroxyl group
Cut at 5' end forms a loop
3' end has acceptor site
Defining a Codon
Gamow
One nucleotide coded for one amino acid
1-base code not enough to code all 20 amino acids
2-base code also not enough
4x1=4
4x4=16
3-base code
4x4x4=64
More than enough, but accommodates 20 amino acids
Standard Code
Codons written in 5'-3' direction
Non-template strand is coding strand
Same as RNA except for U replaces T in RNA
AUG
Start Codon
Methionine
UGG
Tryptophan
Redundancy
Serine
4 triplets
Unique condon triplets
Never codes for more than one amino acid
Stop Codon
do not code
Reading Frames
Open Reading Frame
Continuous sequence of a gene, begins with triplet start codon, ends with triplet stop codon
Inserting Nucleotides
When adding or subtracting nucleotide, it changes codon sequence, resulting in changes to amino acids
Frameshift Mutations
Add or remove 2 nucleotides
Produce Alternate Proteins
First/Second/Third Reading Frames
Coding region of a gene
mRNA can be read from 1st, 2nd, 3rd nucleotide
Removing 3 Nucleotides
No frameshift
Protein still conserved
same for adding 2 nucleotides
RNA Splicing & Spinal Muscular Atrophy
Non-mature mRNA has exon and intron
Splicing Fails
Errors in splicing
Missing sequences
Exon skipping
Intron retention
Not producing full length protein
SMA
Onset before 6mths
Lifespan 2yrs
Affects motor movement
Skeletal muscles die
Neuron degeneration
Autosomal recessive disorder
Chromosome 5
Delta 7
SMN1 Mutations
8 exons (9 technically)
Delta 7 has 7 exons
Missing amino acid
Ribosomes don't translate nucleotide
SMA2
More = longer life expectancy
Backup for SMA1
Splicing
Need high number
Position 6, T
Splicing
Position 6, C
Cut at 7
Skipped at 7
Gene Therapy
Add SMN1
Molecular Therapy
Stop SMN2 exton 7 skipping
Molecular Components of Translation
Process of Translation
Components
Cellular components read genetic message in mRNA,
Translate into primary amino acid
Initiation
Elongation
tRNA
mRNA genetic message to polypeptide
Cytoplasmically situated amino acids -> Transfer amino acids -> Ribosome
Clover-Leaf
Hydrogen bonding between complementary nucleotide bases
4 double-helical segments and 3 loops
L-shape
Fold upon itself
Anticodon
Nucleotide triplet forms complementary base-pair with mRNA codon, coding for amino acid
Aminoacyl tRNA synthetases
Activation of tRNA molecule with amino acid, carried out by enzymes
Enzymes catalyze tRNA to amino acid using ATP energy
Released from enzyme
Grows polypeptide chain on ribosome
Codon-Anticodon Pairing
Correct pairing of tRNA anticodon to mRNA codon
Base-pairing between mRNA codon and tRNA anticodon
Codon-anticodon pairing interactions
Wobble
First base (5') of codon, bind to last base (3')
Flexibility in base pairing between third nucleotide of codon, and tRNA anticodon
Initiation
Eukaryotes
5' cap of mRNA, scans until AUG start codon
Prokaryotes
Shine-Dalgarno sequences
Translation initiation complex assembles at 1+ ribosome binding sites
Polycistronic mRNA
Functionally related genes grouped together
Genes transcribed as single unit
Assembling Initiation Complex
Large & small subunits form functional ribosome when attached to mRNA molecule
Initiation factors bind to 5' cap of mRNA
Initiation factors bind to tRNA, charged with methionine
5'-3' until AUG
Functional Ribosomes
Methionine located in peptidyl (P) site
Charged tRNA enters & binds within aminoacyl (A) site
Peptide Bond Formation
Change in rRNA forms condensation reaction as peptide bond, transfers polypeptide chain into tRNA in A-site
Change in rRNA allowing fo peptidyl-transferase reaction
Ribosome translocates along mRNA
Enabled by binding of GTP-bound elongation factors
Cause deacylated tRNA to move from P-site to exit (E-site)
Termination
Ribosome reaches stop codon
mRNA sequence, GTP-bound release factors bind to A-site
Catalyze hydrolysis of terminal amino acid in polypeptide and tRNA in P-site
GTP hydrolysis dissociates translation complex, ribosomal subunits, tRNA
Genome to Protemone
Complexity
Proteome represents full number of proteins expressed by hereditary information in DNA
Genome
Single genes can encode multiple proteins
Reading, Interpreting, Processing Messages
Double membrane nucleus
Compartmentalization
Control in regulation of cellular processes
Mature mRNA exported out of nucleus
Cytosol
Free or ER bound ribosomes facilitate translation into polypeptides
Interpreting Signal
Cells Detect Changes in Environment
Stimuli resulting in cellular responses
Glucose Absorbed in Small Intestine
Absorbed Glucose Transported to Target Regions
Microvilli cells absorb glucose in intestinal tract
Sensori responses
Glucose absorbed into bloodstream
Micovilli cells
Associated with small blood vessels
Transported to blood vessles
Insulin
mRNA Processing & Protein Isoforms
Alternative Splicing
One pre-mRNA spliced at different junctions resulting in many different mature mRNA molecules
Different combination of transcribed exons
Exons may be excluded from splicing process
Removed
Produces isoforms
Mature mRNA from same pre-mRNA transcript
Regulate gene expression
Insulin
Termination
mRNA isoform translated into higher affinity insulin receptor in muscle cells
Lower blood glucose levels
Absorb glucose to meet high energy needs
Liver cells
Lower affinity insulin
High glucose stimulus detected in pancreas
Insulin is effector signal
Targets body's cells to absorb glucose from bloodstream
Increase glucose absorption
Antibiotics
Block growth & multiplication of microbes
Kill bacteria
Interfering with cell wall synthesis
Disrupting bacterial protein synthesis at ribosome
Inhibiting function of enzymes needed for DNA and RNA synthesis
Targets
tRNA binding sites
mRNA path
A, P, E sites
Tetracycline
Blocking A-site
Interfere with tRNA delivery
Streptomycin
Disrupting tRNA transfer
Prevent transfer of tRNA from A site to P site
Misreading
Brings inappropriate codon to binding site
Large ribosomal subuit
Oxazolidinones
Blocking large subunit
Translation cannot start
Chloramphenicol
Blocking peptide bond formation
Macrolides
Blocking peptide exit tunnel
Erythromycin
Translocation prevented
Move from A-site to P-site
Block P-site
Resistance
Inherited survival mechanism
Drug misuse
Misdiagnoses
Not taking full drug course
Chloramphenicol
rRNA mutations in large ribosomal subunit
Disrupts binding site
Methylation of rRNA prevents binding
Erythromycin
rRNA mutation at tunnel enterance
Add methyl group, block antibiotic binding