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PoL U5 From Gene to Protein

Ch. 17 Campbell's 6th Edition
by

Richard DeLoughery

on 19 January 2016

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Transcript of PoL U5 From Gene to Protein

From Gene to Protein
Section 1 of 3: Connection between Genes and Proteins
Section 2 of 3: Synthesis and Processing of RNA
Section 3 of 3: Synthesis of Protein
Part 1 of 4: Metabolic defects provide evidence
Part 2 of 4: Transcription and Translation
Part 3 of 4: Nucleotide Triplets = Amino Acids
Part 4 of 4: Evolution of Genetic Code
Part 1 of 2: Transcription
Part 2 of 2: Modifying RNA
Part 1 of 6: Translation
Part 2 of 6: Signal Peptides
Part 3 of 6: RNA's multiple roles: Review
Part 4 of 6: Protein synthesis comparison in Pro and Euk: Review
Part 5 of 6: Mutations
Part 6 of 6: What is a Gene?
1909 Archibald Garrod is 1st to suggest genes dictate phenotypes
He used example of alkaptonuria - black urine caused by chemical alkapton (Garrod thought they were missing an enzyme)
Breakthrough showing relationship in 1930's by George Beadle and Edward Tatum
X-rayed bread mold and looked for changes in survivors
Mutants could not survive on minimum diet of wildtype
Could survive on complete medium which supplied all 20 amino acids
Took mutant sample and grew in different vials, each with minimal medium +1 other thing
If mutant grew fine on minimal medium + arginine, it was defective in synthesizing arginine
Then looked more closely at each mutant by doing genetic crosses and realizing that there were 3 types of arginine-lacking mutants, each with their mutation on a different gene.
Suspected that a precursor was converted to ornithine to citrulline to arginine
Grew the 3 different mutations on 4 different mediums
minimal
minimal + ornithine
minimal + citruline
minimal + arginine
Results supported one gene, one enzyme hypothesis = function of a gene is to dictate the production of a specific enzyme
Hypothesis is tweaked over years
not all proteins are enzymes (keratin, insulin)
but some proteins are made of 2 or more polypeptide chains (hemoglobin)
Now can restate as "One gene-one polypeptide hypothesis"
Genes provide instructions for making proteins, don't actually make them
Need RNA to do this
Transcription - synthesis of RNA (any kind) from DNA
DNA acts as template for messenger RNA (mRNA)
Translation - synthesis of a polypeptide from mRNA
mRNA into amino acid sequence at ribosomes
In prokaryotes transcription and translation happen at same time
In eukaryotes, transcription and translation are separated by nuclear membrane
pre-mRNA/primary transcript is modified through RNA processing
4 nucleotides for 20 amino acids
1 nucleotide per amino obviously doesn't work, neither does 2.
3 nucleotides gives 4cubed or 64 possible codes
Experiments verify triplet code used to create polypeptide
Transcription = mRNA made from template strand of DNA
Which strand of DNA used can be different
mRNA is complementary to template strand following same base pairing rules
Remember: No T in RNA, instead U (uracil)
mRNA base triplets called codons
Translation = mRNA codons translated into amino acid sequence; read in 5' to 3' direction along mRNA
1960s experiments show amino acid translations
1961 Marshall Nirenberg used only uracil to make an mRNA strand creating UUU codon
Placed synthesized "poly(U)" in test-tube mix with components required for protein synthesis
Polypeptide formed contained only the amino acid Phenylalanine
So UUU = Phe, other codes figured out by mid-1960s
AUG = start codon and methionine (which may be removed from polypeptide by enzymes later)
3 codons, UAA, UAG, and UGA, are stop codons
Rest of code shows redundancy, but no ambiguity: GAA and GAG = Glutamic acid (redundant), but neither makes any other amino acid (no ambiguity)
3rd base of triplet may be different in redundant codons:
CG_ = Arg,
GG_ = Gly
(more on this later)
Reading frame = correct groupings
UGG UUU GGC UCA if frame is shifted
GGU UUG GCU CA
Genetic code is nearly universal: CCG makes proline in all organisms examined
Few exceptions: some single-celled eukaryotes, mitochondria and chloroplasts
Shared by so many organisms this code must have occurred very early in life's history
RNA polymerase opens DNA strand and attaches RNA nucleotides to each other according to template
Also can only add nucleotides to 3' end
(elongates 5' to 3')
Promoter - DNA sequence that marks where transcription should begin
Terminator - where it should end
(promoter is "upstream" from terminator)
Transcription unit - actual piece DNA that is transcribed
Bacteria have only 1 type of RNA polymerase that synthesize mRNA and other types of RNA as well
Eukaryotes have RNA polymerase I, II, and III (RNA polymerase II used for mRNA synthesis)
3 stages of transcription
1) Initiation
2) Elongation
3) Termination
Initiation
- Promoter typically stretches several dozen nucleotide pairs upstream from start point
- Promoter contains transcription starting point where RNA polymerase binds and determines which strand is template
Prokaryotes - RNA polymerase recognizes and binds to promoter
Eukaryotes - transcription factors (collection of proteins) help the binding of RNA polymerase and initiation of transcription
RNA polymerase + transcription factors = transcription initiation complex
Promoters usually have a TATA box about 25 nucleotides upstream of transcriptional starting point
Once polymerase is attached to promoter, DNA unwinds there and template strand begins to be transcribed
10 - 20 DNA bases are exposed at a time by RNA polymerase; nucleotides added to 3' end of growing RNA strand and DNA closes behind it
Single gene can be transcribed by multiple polymerases allowing for more proteins to be made from that one gene
How transcription termination works is still not fully understood
The polymerase transcribes a DNA terminator sequence
In prokaryotes, transcription stops after that point and polymerase releases RNA and DNA
In eukaryotes, AAUAAA is termination signal in transcribed RNA but continues 10 - 35 nucleotides past this point
mRNA doesn't leave the nucleus directly after transcription but must be modified by enzymes first
Modifications usually include altering the ends and removing interior pieces
5' end (1st end made) is capped with modified guanine nucleotide called a 5' cap
Protects it from hydrolytic enzymes and acts as attachment signal for ribosomes
3' end has a poly(A) tail of 50 - 250 adenines; also protects from enzymes and seems to help strand leave nucleus
Between these caps and the actual mRNA that will be translated, there are leader and trailer segments
In eukaryotes, RNA splicing removes portions of RNA (introns) leaving behind the expressed regions (exons)
Average length of transcribed RNA = 8,000 nucleotides
After RNA splicing = about 1,200 average
These exons are actually broken up by introns, giving "split genes" discovered in 1977 by Richard Roberts and Phillip Sharp
short nucleotide sequences at end of introns are recognized by small nuclear ribonucleoprotein particles (snRNPs = snurps)
several snRNPs and other proteins form a spliceosome which interacts with sequences at end of introns
spliceosome cuts out intron and joins exons together
snRNA may play a role in spliceosome assembly and site recognition but also have a catalytic role
RNA in snRNP = small nuclear RNA
snRNPs are made of RNA and proteins
Ribozymes are RNA molecules that funtion as enzyme
Ribozymes in some sequences may splice themselves out of an RNA strand
Split genes allow for 1 gene to make 2 or more proteins depending on which parts are the exons
This is called alternative RNA splicing
Split genes may allow for evolution of new proteins
Exon shuffling from crossing over can lead to new proteins with new functions
Transfer RNA (tRNA) interprets mRNA code into protein
tRNA transfers amino acids to ribosome
The 20 amino acids are synthesized by cell or absorbed
tRNA have nucleotide triplets that are complementary to a codon on mRNA called anticodons
We'll use prokaryotes as our example
tRNA also transcribed from DNA templates
tRNA molecules are used repeatedly to carry AA's to ribosome
Each tRNA is about 80 nucleotides long and folded into a 3-D structure based on nucleotide interactions
Loop at bottom includes anticodon opposite end has protruding 3' end that attaches to amino acid
Only 45 different tRNA's for the 61 different mRNA codons. How?
Base pairing rules for 3rd base of codon and its anticodon is not as strict as usual. U of tRNA can match with A or G of codon
This is known as wobble and the 3rd position of anticodon is known as the wobble position
Some tRNA have an adenine that has been altered by enzymes and becomes a inosine (I)
Inosine in the wobble position can hydrogen-bond with U, C, or A
As for amino acid - tRNA connections, they are connected by 1 of 20 aminoacyl-tRNA synthetase enzymes (1 for each AA)
Synthetase catalyzes covalent bond between AA and tRNA through hydrolysis of ATP
now called aminoacyl tRNA and it goes to ribosome
Speaking of ribosomes, they are made of 2 subunits (large and small) and are made of proteins and ribosomal RNA (rRNA), the most abundant type of RNA
These subunits are made in the nucleolus as they are transcribed from DNA, processed and put together with proteins
These subunits leave through nuclear pore and become functional when they attach to mRNA
Ribosomes in eukaryotes are larger and have a slightly different molecular structure
Differences can be exploited in antibiotics such as tetracycline which will affect prokaryote ribosomes (How might this be a problem for you also?)
Each ribosome has 1 binding site for mRNA and 3 for tRNA
P site - peptidyl-tRNA
A site - aminoacyl-tRNA
E site - exit site
P site holds the tRNA with AA chain
A site holds tRNA with next AA
E site is where tRNA w/out AA leaves
New AA attaches to carboxyl end of polypeptide
Ribosomes are like 1 large ribozyme since RNA catalyzes the peptide bond formation
So what are the steps of translation? Well, here we go.
Initiation, Elongation, and Termination
All stages require protein factors and for initiation and elongation the hydrolysis of GTP provides energy.
1) Initiation-
small subunit binds to mRNA (leader segment at 5' end) and initiator tRNA (methionine, UAC)
Pro - rRNA of small subunit binds with sequence on mRNA
Euk - 5' cap signals small subunit to attach to 5' end
mRNA, tRNA, and small subunit attach to large subunit = translation initiation complex
Initiation factors bring all of these together
GTP fuels complex formation
Initiator tRNA is in P site, and A is vacant
2) Elongation- (<1/10th of second)
AA are added 1 by 1 and require elongation factors (proteins) and is a 3 step process
I -
Codon Recognition
-
mRNA codon in A site forms H bonds with anticodon of tRNA which is brought by a protein using 2 GTPs
II -
Peptide bond formation
-
rRNA of large subunit catalyzes peptide bond between carboxyl end of polypeptide in P site and AA in A site
Polypeptide seperates from tRNA
III -
Translocation
-
tRNA moves (translocates) to P site (other tRNA moves to E site and exits)
tRNA is still H bonded to mRNA so it comes with it
1 GTP molecule powers this step
mRNA moves into ribosome 5' end first (=ribosome moving 5' -> 3' on mRNA)
3) Termination -
Stop codon is reached (UAA, UGA, and UAG)
Release factor binds to stop codon in A site causing addition of water to polypeptide chain (hydrolyzes it) causing it to split from tRNA
Translation assembly comes apart
1 polypeptide in < 1 minute
Occurs with many ribosomes (polyribosomes)
Polypeptide chain gets folds and bends (gene determines primary structure which determines conformation) - What protein will help these fold properly without outside interferance?
Chaperonins!
Posttranslational Modifcations -
AAs modified by attachment of sugars, lipids, phosphate groups, etc..
Enzymes may remove AA(s)
May be cleaved in 2 or more pieces
Remember 2 types of Ribosomes:
Free - proteins for use in cytosol
Bound - attached to cytosolic side of ER for endomembrane system and secretion
Can switch between the 2 locations
Signal peptide (~20 AA near leading end) marks a polypeptide meant for endomembrane system and tells ribosome to go there
Signal-recognition particle (SRP) is made of RNA and a protein and recognizes signal peptide
SRP brings ribosome to ER receptor protein and then is removed by enzyme
Polypeptide synthesized is either released into cisternal space of ER (secretory) or embedded in ER membrane (membrane protein)
Signal peptides may signal ribosomes to mitochondria, chloroplasts, nucleus interior, and others but translation is completed first
Primary Transcript - RNA before processing
mRNA - messenger RNA
tRNA - transfer RNA
rRNA - ribosomal RNA (ribosomes)
snRNA - small nuclear RNA (spliceosomes)
SRP RNA - signal recognition particles
RNA can H-bond to other nucleic molecules
and can for 3-D shapes based on H-bonds between bases
Differences:
RNA polymerases different
Eukaryotes need transcription factors
Transcription termination done differently
Ribosomes
Transcription/Translation Location
Euks have ability to target organelles
Source of these differences = mutations
Mutations - change in genetic material
Point mutation - change in 1 base pair
In gametes it may be passed to offspring
Bad affect = genetic disorder or hereditary disease
2 Categories:
Base-pair Substitutions or
Base-pair insertions or deletions
Type 1: Substitutions
1 base pair is replaced by another
Silent = no effect on protein
Other substitutions cause obvious changes
May be beneficial, novel, or detrimental
Missense Mutations - causes AA to change to another AA, still makes sense just not the right sense
Nonsense Mutation - causes AA to change to stop codon, premature termination, usually forming a nonfunctional protein
Type 2: Insertions and Deletions
Often much more detrimental to protein
Cause Frameshift Mutations
produce nonfunctional proteins
Mutagens cause change in DNA, can be physical or chemical
Spontaneous Mutations - mutations from errors in DNA replication, repair, or recombination
Hermann Muller (1920's) found x-rays caused fruit fly mutations, must cause humans damage also
Mutagenic Radiation (physical) includes UV
Chemical Mutagens -
Base Analogues - similar to DNA bases but
pair incorrectly
Some Interfere with replication by inserting
themselves
Some change base properties affecting pairing
Mutagens often cause cancer (carcinogens)
After all of this talk of genes we get this definition
"A gene is a region of DNA whose final product in either a polypeptide or an RNA molecule"
http://www.hhmi.org/biointeractive/triplet-code
Spontaneous - no outside influence
Errors with:
DNA Polymerase
Meiosis - nondisjunction, aneuploidy
Gene Sequence disruption
chromosome break, deletions, duplications, inversions, translocations

Induced Mutations - mutagen caused
Chemicals
alter nucleotides
Radiation damage
Ionizing (X-rays & gamma rays) detach e- forming free radicals
Couple of ways to categorize
Somatic Mutation - in body cells and passed to the daughter cells; not passed to offspring
Germline Mutation - in cells that give rise to gametes; passed to offspring
Full transcript