Bio1A Unit 2-6 DNA to Protein Notes File

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Transcript Bio1A Unit 2-6 DNA to Protein Notes File

The Flow of Genetic Information
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The information is in form DNA sequences
DNA leads to traits by dictating protein synthesis
Proteins are the links between genotype and phenotype
Gene expression, the process by which DNA directs protein synthesis,
includes two stages: transcription and translation
Transcription RNA polymerase reads DNA  synthesizes RNA complementary to template DNA
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ribose
GAUC
single-stranded
anti-parallel (RNA occasionally double-stranded)
G:C & A: U
Gene A
Gene X
chromosome
Gene Q
Coding strand
5’-GGAATGCCCCCGTAAC-3’
3’-CCTTACGGGGGCAGGG-5’
Template strand
Transcription
by RNA polymerase
5’-GGA AUG CCC CCG UAA C-3’
mRNA
Translation
by ribosome
Met-Pro-Pro
or rRNA , tRNA, other….
Amino acid sequence
Translation
Ribosomes read the mRNA sequence and synthesizes a polypeptide. The order of the amino
acids is determined by the mRNA sequence which is determined by the DNA sequence.
• Codons - mRNA sequence read in triplets
• Codon order determines amino acid sequence
mRNA codons decoded into amino acids
Starts at 1st AUG (1st amino acid is always Met, However, Met can be used in the middle too)
AUG determines frame (called the reading frame, there are 3 possible reading frames)
Each triplet (codon) represents 1 amino acid
Continues until stop codon = no amino acid
Types of Point Mutations – 2 General categories
Base-pair substitutions
• Silent – mutations, but no change in amino acid sequence
• Missense – single amino acid change
• Nonsens e – altered amino acid is changed into a stop codon
Base-pair insertions or deletions
• Frameshit – all amino acids after mutation are also altered
Genes are more than code for protein
Prokaryotic RNA polymerase binds promoters
Initiation
Terminator
Termination point
Upstream
of gene
Coding Sequence
The three stages of transcription:
1. Initiation (our focus)
• melting and 1st nucleotide
2. Elongation
3. Termination
RNA Polymerase
•separates 2 strands and initiates transcription
•No need for primer
•Uses NTPs to start and build (polymerize) RNA
Eukaryotic Transcription
• Promoters signal the initiation of RNA
synthesis Basal level transcription
• Transcription factors (required) mediate the
binding of RNA polymerase and the initiation
of transcription
• The completed assembly of transcription
factors and RNA polymerase II bound to a
promoter is called a transcription initiation
complex
• A promoter called a TATA box is crucial in
forming the initiation complex in eukaryotes
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Fewer hydrogen bonds – easier to melt
Downstream
Unlike Replication, Transcription bubble does NOT go both ways
After RNA pol moves – DNA behind it renatures
Elongation
Nontemplate
RNA nucleotides
RNA
polymerase
3’
3’ end
5’
Newly
5’
made
RNA
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Direction of
transcription
(“downstream”)
Template
As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at a time
40 nucleotides per second in eukaryotes
A gene can be transcribed simultaneously by several RNA polymerases
In prokaryotes translation can occur simultaneously with transcription due to the lack of a
nuclear membrane
Termination
• Bacteria: polymerase stops
transcription at end of
terminator
• In eukaryotes, the
polymerase continues
transcription after the premRNA is cleaved from the
growing RNA chain; the
polymerase eventually falls
off the DNA
ppp-
AUG
Translation start
UAA
-OH
UAG
UGA
Translation stop
Prokaryote gene/mRNA Structure
Transcription
Initiation
promoter
Transcription
Termination
DNA
5’ untranslated
3’ untranslated
AUG
mRNA
UAA
3’-OH
5’-PPP
Translation Start
Stop
Ribosome binding site
Shine-Delgarno (only in bacteria)
Eukaryotes: Alteration of mRNA Ends
• Each end of a pre-mRNA molecule is modified in a particular way:
– The 5 end receives a modified nucleotide 5 cap
– The 3 end gets a poly-A tail
• These modifications share several functions:
– They seem to facilitate the export of mRNA
– They protect mRNA from hydrolytic enzymes
– They help ribosomes attach to the 5 end
Primary Transcript: or pre-mRNA a.k.a- hnRNA (heteronuclear)
After Transcription, before leaves nucleus
Guanosine
Cap added
G-PPP
Splicing components
• snRNA – small nuclear RNA
• snRNP – small nuclear ribonucleo-protein
complex. Each has one snRNA and about 7
different proteins.
• There are 5 different snRNPs, each with its
own distinct snRNA (U1, U2, U4, U5, or U6)
and distinct proteins.
• Spliceosome – complex of all the snRNPs that
work together to mediate splicing
Importance of Introns
• Some genes can encode more than one kind of
polypeptide, depending on which segments are
treated as exons during RNA splicing
• Such variations are called alternative RNA splicing
• Because of alternative splicing, the number of
different proteins an organism can produce is much
greater than its number of genes
Domains
• Proteins often have a modular architecture
consisting of discrete regions called domains
– can have a isolated function
– Ex: DNA Pol I has a domain for
exonuclease activity and a separate
domain for polymerase activity
• In many cases, different exons code for the
different domains in a protein
• Exon shuffling may result in the evolution of
new proteins
Ribozymes
• Ribozymes are catalytic RNA molecules that function as enzymes and can splice RNA
• The discovery of ribozymes rendered obsolete the belief that all biological catalysts were
proteins
• 3 properties of RNA enable it to function as enzyme
– It can form a three-dimensional structure because of its ability to base pair with itself
– Some bases in RNA contain functional groups
– RNA may hydrogen-bond with other nucleic acid molecules
Molecular Components of Translation
• A cell translates an mRNA message into protein with
the help of transfer RNA (tRNA)
• Molecules of tRNA are not identical:
– Each carries a specific amino acid on one end
– Each has an anticodon on the other end; the
anticodon base-pairs with a complementary
codon on mRNA
•Ribosome has 3 sites where tRNAs bind
•Catalyzes peptide bond formation
•tRNAs anticodon sequence determines
bonding and amino acid
The Structure and Function of Transfer RNA
• A tRNA molecule consists of a single RNA strand that is only about 80 nucleotides long
• Flattened into one plane to reveal its base pairing, a tRNA molecule looks like a cloverleaf
Determines
codon
Single stranded
Intramolecular base
pairing
allows recognition by
aminoacyl-tRNA synthetase
Shape
 aa that is attached to
particular tRNA
• Accurate translation requires two steps:
– First: a correct match between a tRNA and an amino
acid, done by the enzyme aminoacyl-tRNA synthetase
– Second: a correct match between the tRNA anticodon
and an mRNA codon
• Flexible pairing at the third base of a codon is called wobble
and allows some tRNAs to bind to more than one codon
ex: the codon for threonine is always ACx with the 3rd
position variable. A single tRNA can suffice with UGI
where I is Ionisine (a modified adenine). Ionisine can
base pair with multiple bases.
•tRNA for each amino acid have own shape
ex: all Threonine tRNAs have same shape
•For each kind of tRNA are therefore all
recognized by the same aminoacyl-tRNA
synthetase which will attach its amino acid
ex: all theonine tRNAs will be recognized by
threonine tRNA synthetase and have thr
attached to them
Aminoacyl-tRNA
(“charged tRNA”)
Aminoacyl-tRNA
synthetase
(enzyme)
Ribosomes
• Ribosomes facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis
• The two ribosomal subunits (large and small) are made of proteins and ribosomal RNA (rRNA)
Ribosomal proteins and rRNAs are
encoded by genes rRNAs are NOT
translated though
rRNA gives ribosome shape
Large subunit
• 31 proteins
• 2 rRNA
23S (~3000nt’s)
5S (~300 nt’s)
Enzymatic
Small subunit
• 20 Proteins
• rRNA
16S (~1500 nt’s)
Forms bottom of A P E sites
 Where codons and anticodons interact
Initiation
1. Prokaryotes: Shine-delgarno sequence orients small subunit just prior to AUG
• 1 mRNA transcript can have multiple SD sequences
• In other words, 1 promoter  1 mRNA  multiple genes / gene products / proteins
• Called the operon system: 1 promoter for many genes
1. Eukaryotes: 5’ Cap facilitaties binding of small subunit
A ribosome has three binding sites for tRNA:
• The P site holds the tRNA that carries the
growing polypeptide chain
• The A site holds the tRNA that carries the
next amino acid to be added to the chain
• The E site is the exit site, where discharged
tRNAs leave the ribosome
2. Met-tRNA binds (tenuous)
3. Large subunit attaches (Takes Energy)
Elongation
1. Codon
Recognition
3. Translocation
2. Peptide bond
formation
Termination
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stop codon in A site
The A site accepts a protein called a release factor
causes the addition of a H2O instead of amino acid (Hydolysis)
This reaction releases the polypeptide, and the translation assembly then comes apart