chapter 17 rna and protein synthesis. transcription and translation ...
TRANSCRIPT
Chapter 17RNA and Protein Synthesis
Transcription and Translation
• http://www.dnatube.com/video/3059/DNA-Transcription-and-Protein-Assembly
Overview• Nucleotide sequences responsible for
information on DNA• The DNA inherited yields specific traits by
dictating the synthesis of proteins• Proteins are the links between genotype and
phenotype• Gene expression, the process by which DNA
directs protein synthesis, includes two stages: transcription and translation
History of Gene Expression
• 1909 – Archibald Garrod– First to say genes determine phenotypes through
enzymes that catalyze specific chemical reactions– “inborn errors of metabolism” - symptoms of an
inherited disease reflect an inability to synthesize a certain enzyme
History of Gene Expression
• George Beadle and Edward Tatum – exposed bread mold to X-rays
• mutants were unable to survive on minimal medium – unable to synthesize certain molecules
– Identified three classes of arginine-deficient mutants, each lacking a different enzyme necessary for synthesizing arginine
• One gene–one enzyme hypothesis: states that each gene dictates production of a specific enzyme
More Recent History of Gene Expression
• Many proteins are composed of several polypeptides, each of which has its own gene
• Beadle and Tatum’s hypothesis is now restated as the one gene–one polypeptide hypothesis
The Structure of RNA
• single stranded• working copy of one
gene– Disposable
• RNA is the intermediate between genes and the proteins for which they code
The Structure of RNA
• one gene from DNA can produce many strands of RNA
• instead of thymine (T), uracil (U)
• RNA has A, U, C, G
Transcription and Translation• Transcription is the synthesis of RNA under
the direction of DNA– nucleus
• Transcription produces messenger RNA (mRNA)
• Translation is the synthesis of a polypeptide, which occurs under the direction of mRNA– cytoplasm
• Ribosomes are the sites of translation
Transcription and Translation Overview
• In prokaryotes, mRNA produced by transcription is immediately translated without more processing
• In a eukaryotic cell, the nuclear envelope separates transcription from translation
• Eukaryotic RNA transcripts are modified through RNA processing to yield finished mRNA
Fig. 17-3
TRANSCRIPTION
TRANSLATION
DNA
mRNARibosome
Polypeptide
(a) Bacterial cell
Nuclearenvelope
TRANSCRIPTION
RNA PROCESSINGPre-mRNA
DNA
mRNA
TRANSLATION Ribosome
Polypeptide
(b) Eukaryotic cell
Fig. 17-3a-1
TRANSCRIPTIONDNA
mRNA
(a) Bacterial cell
Fig. 17-3a-2
(a) Bacterial cell
TRANSCRIPTIONDNA
mRNA
TRANSLATIONRibosome
Polypeptide
Fig. 17-3b-1
(b) Eukaryotic cell
TRANSCRIPTION
Nuclearenvelope
DNA
Pre-mRNA
Fig. 17-3b-2
(b) Eukaryotic cell
TRANSCRIPTION
Nuclearenvelope
DNA
Pre-mRNARNA PROCESSING
mRNA
Fig. 17-3b-3
(b) Eukaryotic cell
TRANSCRIPTION
Nuclearenvelope
DNA
Pre-mRNARNA PROCESSING
mRNA
TRANSLATION Ribosome
Polypeptide
17.2 Transcription
DNA RNA
Types of RNA
• three types– messenger RNA
(mRNA)– ribosomal RNA (rRNA)– transfer RNA (tRNA)
Transcription• requires enzyme RNA
polymerase• RNA polymerase binds to
DNA and separates the strands – Similar to DNA replication
Transcription
• RNA polymerase uses one strand of DNA as a template to make RNA
• RNA is made in the 5’ – 3’ direction– How is the DNA
read?
Transcription
• The DNA sequence where RNA polymerase attaches is called the promoter
• In bacteria, the sequence signaling the end of transcription is called the terminator
• The stretch of DNA that is transcribed is called a transcription unit
Fig. 17-7
Promoter Transcription unit
Start pointDNA
RNA polymerase
5533
Initiation1
2
3
5533
UnwoundDNA
RNAtranscript
Template strandof DNA
Elongation
RewoundDNA
5
55
5
5
33
3
3
RNAtranscript
Termination
5533
35Completed RNA transcript
Newly madeRNA
Templatestrand of DNA
Direction oftranscription(“downstream”)
3 end
RNApolymerase
RNA nucleotides
Nontemplatestrand of DNA
Elongation
Fig. 17-7a-1Promoter Transcription unit
DNAStart point
RNA polymerase
553
3
Fig. 17-7a-2Promoter Transcription unit
DNAStart point
RNA polymerase
553
3
Initiation
33
1
RNAtranscript
5 5
UnwoundDNA
Template strandof DNA
Fig. 17-7a-3Promoter Transcription unit
DNAStart point
RNA polymerase
553
3
Initiation
33
1
RNAtranscript
5 5
UnwoundDNA
Template strandof DNA
2 Elongation
RewoundDNA
5
5 5 3 3 3
RNAtranscript
Fig. 17-7a-4Promoter Transcription unit
DNAStart point
RNA polymerase
553
3
Initiation
33
1
RNAtranscript
5 5
UnwoundDNA
Template strandof DNA
2 Elongation
RewoundDNA
5
5 5 3 3 3
RNAtranscript
3 Termination
5
5 5 33
3Completed RNA transcript
Fig. 17-7b
Elongation
RNApolymerase
Nontemplatestrand of DNA
RNA nucleotides
3 end
Direction oftranscription(“downstream”) Template
strand of DNA
Newly madeRNA
3
5
5
Transcription Overview
• http://www.youtube.com/watch?v=WsofH466lqk
Three Stages of Transcription
• Initiation
• Elongation
• Termination
Initiation
• Signaled by promoters• Transcription factors mediate the binding of
RNA polymerase and the initiation of transcription
• Transcription initiation complex: all transcription factors and RNA polymerase II bound to a promoter
• A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes
Fig. 17-8A eukaryotic promoterincludes a TATA box
3
1
2
3
Promoter
TATA box Start point
Template
TemplateDNA strand
535
Transcriptionfactors
Several transcription factors mustbind to the DNA before RNApolymerase II can do so.
5533
Additional transcription factors bind tothe DNA along with RNA polymerase II,forming the transcription initiation complex.
RNA polymerase IITranscription factors
55 53
3
RNA transcript
Transcription initiation complex
Elongation
• RNA polymerase untwists the DNA, 10 to 20 bases at a time
• Transcription progresses at a rate of 40 nucleotides per second in eukaryotes
• A gene can be transcribed simultaneously by several RNA polymerases
Termination
• Bacteria: the polymerase stops transcription at the end of the terminator
• Eukaryotes: the polymerase continues transcription after the pre-mRNA is cleaved from the growing RNA chain– Pre-mRNA will be cleaved when the RNA
polymerase II transcribes a polyadenylation sequence (AAUAAA on the pre-mRNA)
– the polymerase eventually falls off the DNA
17.3 RNA Processing
RNA Processing
• Enzymes in the eukaryotic nucleus modify pre-mRNA
• During RNA processing, both ends of the primary transcript are usually altered
• Some interior parts of the molecule are cut out, and the other parts spliced together
RNA Modifications: the ends
• Each end of a pre-mRNA molecule is modified in a particular way:– The 5’ end receives a modified guanine nucleotide
5’ cap– The 3’ end gets a poly-A tail (made of 50-250 more
adenine nucleotides)
RNA Modifications: the ends
• These modifications share several functions:– Facilitate the export of mRNA– Protection of the mRNA from hydrolytic enzymes– They help ribosomes attach to the 5’ end
Fig. 17-9
Protein-coding segment Polyadenylation signal3
3 UTR5 UTR
5
5 Cap Start codon Stop codon Poly-A tail
G P PP AAUAAA AAA AAA…
RNA Splicing
• Noncoding stretches of nucleotides that lie between coding regions• intervening sequences or introns
• Exons: eventually expressed, translated into amino acid sequences
• RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence
RNA Processing
• RNA will copy both introns and exons from the DNA– Introns need to be cut out
to make a working piece of RNA
• Remaining pieces of exons will be spliced together to form the final mRNA sequence
Fig. 17-10
Pre-mRNA
mRNA
Codingsegment
Introns cut out andexons spliced together
5 Cap
Exon Intron5
1 30 31 104
Exon Intron
105
Exon
146
3Poly-A tail
Poly-A tail5 Cap
5 UTR 3 UTR1 146
• In some cases, RNA splicing is carried out by spliceosomes
• Spliceosomes consist of a variety of proteins and several small nuclear ribonucleoproteins (snRNPs) that recognize the splice sites
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 17-11-1RNA transcript (pre-mRNA)
Exon 1 Exon 2Intron
ProteinsnRNA
snRNPs
Otherproteins
5
Fig. 17-11-2RNA transcript (pre-mRNA)
Exon 1 Exon 2Intron
ProteinsnRNA
snRNPs
Otherproteins
5
5
Spliceosome
Fig. 17-11-3RNA transcript (pre-mRNA)
Exon 1 Exon 2Intron
ProteinsnRNA
snRNPs
Otherproteins
5
5
Spliceosome
Spliceosomecomponents
Cut-outintronmRNA
Exon 1 Exon 25
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
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
RNA can function as an 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
Alternative RNA Splicing
• Some genes can encode more than one kind of polypeptide, – depending on which segments are treated as
exons during RNA splicing• Number of different proteins an organism can
produce is much greater than its number of genes
Protein Domains
• Proteins often have a modular architecture consisting of discrete regions called domains
• In many cases, different exons code for the different domains in a protein
• Exon shuffling may result in the evolution of new proteins– Protein will fold differently = act differently
Reading the Genetic Code
The Genetic Code
• Genetic code, or language of mRNA instructions, is read three letters at a time
The Genetic Code
• Each three letter sequence, or “word”, codes for one amino acid• these three letter words are known as codons• Codons are found on the mRNA
• The protein is determined by the order of each amino acid in the sequence
The Genetic Code• There are 64 possible
codons• One codon, AUG, is known
as the “start” codon for protein synthesis
• There are also three possible sequences that are called “stop” codons– Show where polypeptide ends
Translation
Fig. 17-13
Polypeptide
Ribosome
Aminoacids
tRNA withamino acidattached
tRNA
Anticodon
TrpPhe Gly
Codons 35
mRNA
Translation
• Making a protein from a strand of mRNA
tRNA
• 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
Fig. 17-14
Amino acidattachment site
3
5
Hydrogenbonds
Anticodon
(a) Two-dimensional structure
Amino acidattachment site
5
3
Hydrogenbonds
3 5AnticodonAnticodon
(c) Symbol used in this book(b) Three-dimensional structure
In order to have accurate translation…
• 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
Fig. 17-15-1
Amino acid Aminoacyl-tRNAsynthetase (enzyme)
ATP
AdenosineP P P
Fig. 17-15-2
Amino acid Aminoacyl-tRNAsynthetase (enzyme)
ATP
AdenosineP P P
AdenosineP
PP i
PPi
i
Fig. 17-15-3
Amino acid Aminoacyl-tRNAsynthetase (enzyme)
ATP
AdenosineP P P
AdenosineP
PP i
PPi
i
tRNA
tRNA
Aminoacyl-tRNAsynthetase
Computer model
AMPAdenosineP
Fig. 17-15-4
Amino acid Aminoacyl-tRNAsynthetase (enzyme)
ATP
AdenosineP P P
AdenosineP
PP i
PPi
i
tRNA
tRNA
Aminoacyl-tRNAsynthetase
Computer model
AMPAdenosineP
Aminoacyl-tRNA(“charged tRNA”)
Ribosomes
• Ribosomal RNA (rRNA)• Large subunit• Small subunit
Fig. 17-16Growingpolypeptide Exit tunnel
Largesubunit
Smallsubunit
tRNAmolecules
E P A
mRNA5 3
(a) Computer model of functioning ribosome
P site (Peptidyl-tRNAbinding site)
E site(Exit site)
A site (Aminoacyl-tRNA binding site)
E P A Largesubunit
mRNAbinding site
Smallsubunit
(b) Schematic model showing binding sites
Amino end Growing polypeptide
Next amino acidto be added topolypeptide chain
mRNAtRNAE
3
5 Codons
(c) Schematic model with mRNA and tRNA
Fig. 17-16b
P site (Peptidyl-tRNAbinding site) A site (Aminoacyl-
tRNA binding site)E site(Exit site)
mRNAbinding site
Largesubunit
Smallsubunit
(b) Schematic model showing binding sites
Next amino acidto be added topolypeptide chain
Amino end Growing polypeptide
mRNAtRNA
E P A
E
Codons
(c) Schematic model with mRNA and tRNA
5
3
Ribosomes
• A ribosome has three binding sites for tRNA:– The P site (peptidyl-tRNA binding site) holds the
tRNA that carries the growing polypeptide chain– The A site (Aminoacyl-tRNA binding site) holds
the tRNA that carries the next amino acid to be added to the chain
– The E site (exit site) is where discharged tRNAs leave the ribosome
Three Parts of Translation
• Initiation• Elongation• Termination
Steps of Translation
1. mRNA attaches to a ribosome
• a small ribosomal subunit binds with mRNA and a special initiator tRNA
• small subunit moves along the mRNA until it reaches the start codon (AUG)
• initiation factors bring in the large subunit that completes the translation initiation complex
Fig. 17-17
3355U
UA
ACGMet
GTP GDPInitiator
tRNA
mRNA
5 3Start codon
mRNA binding site
Smallribosomalsubunit
5
P site
Translation initiation complex
3
E A
Met
Largeribosomalsubunit
Elongation
• Amino acids are added one by one to the preceding amino acid
• Each addition involves proteins called elongation factors and occurs in three steps:– codon recognition
– peptide bond formation
– Translocation
Elongation: Codon Recognition
2. tRNA brings the correct amino acid to the ribosome
• each tRNA carries only one type of amino acid– the anticodon sequence
is complimentary to the codon
Elongation: Peptide Bond Formation
3. Ribosome forms a peptide bond between the amino acids (forming a protein)
Elongation: Translocation
4. The tRNA from the first amino acid is then released from the ribosome via the E site
5. Ribosome then moves to the next codon and a new tRNA enters the A site
Translation Continues!
6. Polypeptide chain grows until the ribosome reaches a “stop” codon
Termination occurs when a stop codon in the mRNA reaches the A site of the ribosome
Translation Complete!
7. The A site accepts a protein called a release factor
The release factor causes the addition of a water molecule instead of an amino acid
Releases the polypeptide, and the translation assembly then comes apart
Fig. 17-19-1
Releasefactor
3
5Stop codon(UAG, UAA, or UGA)
Fig. 17-19-2
Releasefactor
3
5Stop codon(UAG, UAA, or UGA)
5
3
2
Freepolypeptide
2 GDP
GTP
Fig. 17-19-3
Releasefactor
3
5Stop codon(UAG, UAA, or UGA)
5
3
2
Freepolypeptide
2 GDP
GTP
5
3
Polyribosomes
• Many ribosomes translate a single mRNA simultaneously, forming a polyribosome (or polysome)
• Polyribosomes enable a cell to make many copies of a polypeptide very quickly
Fig. 17-20
Growingpolypeptides
Completedpolypeptide
Incomingribosomalsubunits
Start ofmRNA(5 end)
Polyribosome
End ofmRNA(3 end)
(a)
Ribosomes
mRNA
(b) 0.1 µm
• Polypeptide chains are modified after translation
• Completed proteins are targeted to specific sites in the cell
• During and after synthesis, a polypeptide chain spontaneously coils and folds into its three-dimensional shape
• Proteins may also require post-translational modifications before doing their job
• Some polypeptides are activated by enzymes that cleave them
• Other polypeptides come together to form the subunits of a protein
Ribosomes• Free ribosomes: in cytosol• Bound ribosomes: on surface of ER– Make proteins to be excreted from the cell– All transcription occurs in the cytoplasm• Polypeptides destined for the ER or for secretion are
marked by a signal peptide • A signal-recognition particle (SRP) binds to the signal
peptide• The SRP brings the signal peptide and its ribosome to the ER
Mutations
• Mutations are changes in the genetic material of a cell or virus
• Point mutations are chemical changes in just one base pair of a gene– Substitution– Insertion or Deletion
Effects of Substitutions
• Silent mutations have no effect on the amino acid produced by a codon because of redundancy in the genetic code
• Missense mutations still code for an amino acid, but not necessarily the right amino acid
• Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein
Effects of Insertions and Deletions
• More harmful than substitutions• Insertion or deletion of nucleotides may alter
the reading frame, producing a frameshift mutation
Mutagens
• Spontaneous mutations can occur during DNA replication, recombination, or repair
• Mutagens are physical or chemical agents that can cause mutations
Gene Expression in Archae, Bacteria, Eukaryotes
• Use your text book to compare and contrast the cellular processes of gene expression in archaea, bacteria and eukarya
Genes and Proteins
• genes code for proteins which are what carry out expression of these genes
• proteins code for enzymes which cause certain reactions to take place– these reactions are what cause traits!
Protein Structure
Protein Structure
• Primary (1°)structure: amino acid sequence from translation
• Secondary (2°) structure: alpha (α) helix and beta (β) sheets
Protein Structure
• Tertiary (3º) structure: three dimensional structure– Each protein has its own
unique tertiary structure
Protein Structure• Quaternary (4°)
structure: multiple tertiary structures form a complex
• The protein will change confirmation (shape) if not being used