2010-Chapter 16 - Transcription and Translation-Students

Generating information from genes
Transcription and translation Chapter 16
Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley
Introduction
A cell builds the proteins it needs from instructions encoded in its genome.
The flow of information in the cell is as follows:
Transcription in Bacteria
Transcription
RNA polymerase performs this synthesis by transcribing only one strand of DNA, called the template strand. The other DNA strand is called the non-template strand. The sequence of the non-template strand matches the sequence of the RNA, except that RNA has uracil (U) in place of thymine (T). Transcription consists of three phases:
Transcription Is the Synthesis of RNA from a DNA Template

Non-template (coding) strand 5 5 3 Template strand 3 5 DNA 3 RNA 3 5
Phosphodiester bond is formed by RNA polymerase after base pairing occurs
RNA 5
Hydrogen bonds form between complementary base pairs
DNA template
RNA Polymerase Structure and Function
Bacterial RNA polymerase is a large, globular enzyme with several interior channels.
A holoenzyme Prokaryotic RNA polymerase is a holoenzyme made up of the core enzyme, which has the ability to synthesize RNA, and a sigma subunit
The 3D Structure of RNA Polymerase Bound to DNA

Sigma DNA
Holoenzyme
Core enzyme
Initiation: How Does Transcription Begin in Bacteria?
Transcription of bacterial genes is initiated at specific sections of DNA called

Promoters have two key regions: the 10 box and the 35 box. The names come from their locations: for example, the 10 box is found ten bases upstream from the transcription start site. (Downstream is in the direction RNA polymerase moves during transcription; upstream is in the opposite direction.) The sigma subunit is required for the initiation phase of transcription.
Transcription initiation site?

Figure 16-3-1
Promoter (on non-template strand) 35 box

Upstream DNA
10 box
+1 site Sigma
Active site
Downstream DNA
RNA polymerase
1. Initiation begins
Sigma binds to promoter region of DNA.
Initiation: How Does Transcription Begin in Bacteria?
Next, sigma opens the DNA double helix and the template strand is threaded through the RNA polymerase active site.
An incoming ribonucleoside triphosphate (NTP) pairs with a complementary base on the DNA template strand, and RNA polymerization begins.
In Bacteria, Sigma Plays a Key Role in Initiating Transcription

Figure 16-3-2
HOW TRANSCRIPTION BEGINS

Template strand
+1 site
+1 site
Non-template strand
RNA
RNA NTPs
2. Initiation continues
Sigma opens the DNA helix; transcription begins.
In Bacteria, Sigma Plays a Key Role in Initiating Transcription

Figure 16-3-3
HOW TRANSCRIPTION BEGINS

Upstream DNA RNA
RNA exit site
Zipper Rudder
Downstream DNA
3. Initiation is complete
Sigma releases; mRNA synthesis continues.
Elongation and Termination
During the elongation phase of transcription, RNA polymerase moves along the DNA template and synthesizes RNA in the 5' 3' direction.
Transcription ends with a termination phase. In this phase, RNA polymerase encounters a transcription termination signal in the DNA template. This signal codes for RNA forming a hairpin structure.
Transcription Terminates When a Hairpin Forms in the RNA

Figure 16-4-1
HOW TRANSCRIPTION ENDS

Upstream DNA
RNA Hairpin loop

RNA
RNA polymerase
Transcription termination signal
Downstream DNA
1. RNA polymerase reaches a transcription

termination signal, which codes for RNA that forms a hairpin.
Transcription Terminates When a Hairpin Forms

Figure 16-4-2
HOW TRANSCRIPTION ENDS

RNA
DNA
2. The RNA hairpin causes the RNA strand

to separate from the RNA polymerase, terminating transcription.
Transcription and RNA Processing in Eukaryotes
Eukaryotic transcription shares many fundamental characteristics with prokaryotic transcription.

Transcription in eukaryotes is initiated by proteins called basal transcription factors. These factors begin transcription by matching RNA polymerase with the appropriate promoter region in DNA, a function analogous to that of sigma in bacteria. Eukaryotic cells contain three types of RNA polymerase, named I, II, and III. Each of these enzymes transcribes different classes of RNA.
Transcription and RNA Processing in Eukaryotes
The promoters in eukaryotic RNA are more diverse and complex than are bacterial promoters.
The promoters recognized by each type of RNA polymerase differ. Many promoters recognized by RNA polymerase II include a sequence called a TATA box analogous in function to the prokaryotic 10 and 35 boxes. In eukaryotes, transcription is followed by several important RNA processing steps.
The Startling Discovery of Eukaryotic Genes
The RNA made in the nucleus was much longer than the mRNA molecules found in the cytoplasm ,
Seems the protein-coding regions of eukaryotic genes are interrupted by noncoding regions. Exons are the coding regions of eukaryotic genes that will be part of the final mRNA product. The intervening noncoding sequences are called introns, and are not in the final mRNA. Therefore, Eukaryotic genes
Processing of Eukaryote RNA transcripts
Intron 1 Intron 2 DNA 3 5 Exon 1
Promoter
Exon 2
Exon 3 3
Primary RNA transcript 5 Spliced transcript 5 3
Exons, Introns, and RNA Splicing
The transcription of eukaryotic genes by RNA polymerase generates a primary RNA transcript that contains exons and introns. Introns are removed by splicing.
Small nuclear ribonucleoproteins (snRNPs, pronounced snurps) form a complex called a spliceosome.
Introns Are Spliced Out of the Original mRNA

Figure 16-7b
snRNPs ARE THE EDITORS.
Primary RNA 5
A
snRNPs 3 Exon 2
Exon 1
Intron
1. Several snRNPs and

A 5 Spliceosome 3 proteins assemble to form a spliceosome. The 2 hydroxyl group on an adenine nucleotide (A) reacts with the 5 end of the intron, breaking RNA.
2. The 5 end of the

5 5 3 A 5 3 intron becomes attached to the A nucleotide, forming a loop of RNA. The free 3 end of one exon reacts with the 5 end of the other.
Excised intron A
3. The 3 and 5 ends

of adjacent exons bond covalently, releasing the intron (which is then degraded).
Exon 1 Exon 2
3 Mature mRNA
Additional processing - Adding Caps and Tails to RNA Transcripts
Primary RNA transcripts are also processed by the addition of a 5' cap and a poly (A) tail.
With the addition of cap and tail, processing is complete; the product is a mature mRNA. The 5' cap serves as a The poly (A) tail extends the life of an mRNA by protecting it from degradation.
In Eukaryotes, mRNAs Are Given a Cap and a Tail

5 cap
Poly(A) tail 3
5
5 untranslated region
Coding region
3 untranslated region
Translation
In translation, the sequence of bases in the mRNA is converted to an amino acid sequence in a protein.
Ribosomes Are the Site of Protein Synthesis
Ribosomes catalyze translation of the mRNA sequence into protein.

In bacteria, transcription and translation can occur simultaneously. Bacterial ribosomes begin translating an mRNA before RNA polymerase has finished transcribing it. In eukaryotes, transcription and translation are separated. mRNAs are synthesized and processed in the nucleus and transported to the cytoplasm for translation by ribosomes.
Transcription and Translation Occur Simultaneously in Bacteria

5 end
of mRNA
Ribosome translates mRNA as it is being synthesized by RNA polymerase
Protein 1 1 1 Ribosome RNA polymerase Start of gene (3 end of template strand) End of gene (5 end of template strand)
Transcription and Translation Are Separated in Space and Time in Eukaryotes

Figure 16-10
mRNA DNA Transcription and RNA processing in nucleus Mature mRNA
Mature mRNA Ribosome Translation in cytoplasm Protein
How Does an mRNA Triplet Specify an Amino Acid?

Figure 16-11
Hypothesis 1: Amino acids interact directly with mRNA codons.

Peptide bond Amino acids mRNA Codon Codon Codon Codon
Hypothesis 2: Adapter molecules hold amino acids and interact with mRNA codons.
Amino acids
Adapter molecules mRNA Codon Codon Codon Codon
The Role of Transfer RNA
The adapter molecule was later found to be a small RNA called transfer RNA (tRNA).
A tRNA covalently linked to its corresponding amino acid is called an Enzymes called aminoacyl tRNA synthetases.
HOW AMINO ACIDS ARE LOADED ONTO tRNAs

ATP
1. Active site on aminoacyl
tRNA synthetase binds ATP and amino acid. Each aminoacyl tRNA synthetase is specific to one amino acid.
Aminoacyl tRNA synthetase specific to leucine
2. Reaction leaves AMP and

Activated enzyme complex amino acid bound to enzyme; two phosphate groups released. Activated amino acid has high potential energy.
HOW AMINO ACIDS ARE LOADED ONTO tRNAs

tRNA specific to leucine
3. The activated amino acid is

transferred from tRNA synthetase to the tRNA specific to that amino acid; AMP leaves.
4. The finished aminoacyl

tRNA is ready to participate in translation. Aminoacyl tRNA
The Role of Transfer RNA
For each of the 20 amino acids, there is a different aminoacyl tRNA synthetase and one or more tRNAs.
Each tRNA.
Experimental Evidence that Amino Acids Are Transferred from tRNAs to Proteins
What Do tRNAs Look Like?
tRNAs have a cloverleaf
structure.
The CCA sequence at the 3' end of each tRNA is the The triplet loop at the opposite end of the cloverleaf is the anticodon that base pairs with the mRNA codon. The cloverleaf structure of tRNA folds over to produce a molecule with an L-shaped tertiary structure. .
Secondary structure of tRNA

3
Early model of aminoacyl tRNA function

Amino acid 3 Binding site for amino acid
Serine anticodon 5 mRNA
Binding site for mRNA codon 3 Serine codon
Revised model incorporating tertiary structure of tRNA

5 3
Anticodon
Codon 5 mRNA
How Many tRNAs Are There?
There are 61 different codons but only about 40 tRNAs in most cells.
To resolve this deficit, Francis Crick proposed the wobble hypothesis. This hypothesis proposes that the anticodon of tRNAs can still bind successfully to a codon whose third position requires a nonstandard base pairing. Thus, one tRNA is able to base pair with more than one type of codon.
Ribosomes and the Mechanism of Translation
Ribosomes contain both protein and ribosomal RNA (rRNA).

Ribosomes can be separated into two subunits, the large subunit and the small subunit.
Ribbon model of ribosome during translation
The Structure of the Ribosome during Translation

Diagram of ribosome during translation
Polypeptide grows in amino to carboxyl direction (amino acids in green) Peptide bond formation occurs here
Large subunit
Large subunit
Aminoacyl tRNA
mRNA 5 Small subunit Small subunit Codon
Anticodon
tRNA in E site (blue)
tRNA in P site tRNA in A site (green) (red)
The E site holds a tRNA that will exit
The P site holds the tRNA with growing polypeptide attached
The A site holds an aminoacyl tRNA
All three tRNAs are bound at their anticodons to the corresponding mRNA codon.
The A site of the ribosome is the acceptor site for an aminoacyl tRNA. The P site is where a peptide bond forms that adds an amino acid to the growing polypeptide chain. The E site is where tRNAs no longer bound to an amino acid exit the ribosome.
Translation occurs by a three-step sequence:

(1) An aminoacyl tRNA carrying the correct anticodon for the mRNA codon enters the A site; (2) A peptide bond forms between the amino acid on the aminoacyl tRNA in the A site and the growing polypeptide on the tRNA in the P site; (3) The ribosome moves ahead three bases and all three tRNAs move down one position; the tRNA in the E site exits.
Initiation
The initiation phase of translation begins at the AUG start codon.

In bacteria, the start codon is preceded by a ribosome binding site (also called the ShineDalgarno sequence) that is complementary to a section of one rRNA in the small ribosomal subunit. The interaction between the small subunit and the mRNA is mediated by initiation factors.
INITIATING TRANSLATION IN BACTERIA

Ribosome binding site 5 Start codon 3
Initiation factor
Initiation factor
Small subunit of ribosome
1. Ribosome binding site sequence binds

to a complementary sequence in an RNA molecule in the small subunit of the ribosome, with the help of protein initiation factors.
Aminoacyl tRNA
5 Start codon Small subunit of ribosome
2. Initiator aminoacyl tRNA binds

to start codon.
Large subunit of ribosome
3. Large subunit of ribosome binds.

Translation begins.
Initiation
Once the small ribosomal subunit is bound to the mRNA, the initiator aminoacyl tRNA binds to the AUG sequence.
The large subunit binds and completes the initiation complex.
Elongation
At the start of the elongation phase, the initiator tRNA is in the P site, and the E and A sites are empty.
An aminoacyl tRNA binds to the codon in the A site via complementary base pairing between anticodon and codon. .
ELONGATION OF POLYPEPTIDES DURING TRANSLATION
Ribosome
Peptidyl site
tRNA
Exit site mRNA 5
Aminoacyl site
3
Start codon
1. Incoming aminoacyl tRNA

New tRNA moves into A site, where its anticodon base pairs with the mRNA codon.
2. Peptide bond formation

The amino acid attached to the tRNA in the P site is transferred to the tRNA in the A site.
Moving Down the mRNA
After peptide bond formation, the polypeptide on the tRNA in the P site is transferred to the tRNA in the A site.
The ribosome translocates down the mRNA by three nucleotides, and the tRNA attached to the growing protein moves into the P site.
3. Translocation
Ribosome moves down mRNA. The tRNA attached to polypeptide chain moves into P site. The A site is empty.
The A site is now available to accept a new aminoacyl tRNA for binding to the next codon.
4. Incoming aminoacyl tRNA

New tRNA moves into A site, where its anticodon base pairs with the mRNA codon.
5. Peptide bond formation

The polypeptide chain attached to the tRNA in the P site is transferred to the aminoacyl tRNA in the A site.
The tRNA that was in the P site moves to the E site, and if the E site is occupied, that tRNA is ejected.

Exit tunnel Elongation cycle continues
6. Translocation
Ribosome moves down mRNA. The tRNA attached to polypeptide chain moves into P site. Empty tRNA from P site moves to E site, where tRNA is ejected. The A site is empty again.
Is the Ribosome an Enzyme or a Ribozyme?
The active site of the ribosome is entirely ribosomal RNA.

Thus, ribosomal RNA catalyzes peptide bond formation and the ribosome is a ribozyme.
Elongation
Polyribosomes, strings of translating ribosomes, assemble along an mRNA to increase the rate of protein production.
Growing polypeptides mRNA

5 3
Ribosomes
Polyribosomes
Termination
The termination phase starts when the A site encounters a.

This causes a protein called a release factor to enter the site. These factors catalyze hydrolysis of the bond linking the tRNA in the P site with the polypeptide chain.
TERMINATION OF TRANSLATION
Hydrolysis of bond linking tRNA and polypeptide
tRNA mRNA 5 STOP codon
Release factor
1. When translocation opens the A site

and exposes one of the stop codons, a protein called a release factor fills the A site. The release factor catalyzes the hydrolysis of the bond linking the tRNA in the P site with the polypeptide chain.
mRNA 5
2. The hydrolysis reaction frees the

polypeptide, which is released from the ribosome. The empty tRNAs are released either along with the polypeptide or
Large subunit
5 mRNA
Small subunit
3. when the ribosome separates from the

mRNA, and the two ribosomal subunits dissociate. The subunits are ready to attach to the start codon of another message and start translation anew.
Hydrolysis of bond linking tRNA and polypeptide
tRNA mRNA 5 STOP codon
Release factor
1. When translocation opens the A site

and exposes one of the stop codons, a protein called a release factor fills the A site. The release factor catalyzes the hydrolysis of the bond linking the tRNA in the P site with the polypeptide chain.
mRNA 5
2. The hydrolysis reaction frees the

polypeptide, which is released from the ribosome. The empty tRNAs are released either along with the polypeptide or
Large subunit
5 mRNA
Small subunit
3. when the ribosome separates from the

mRNA, and the two ribosomal subunits dissociate. The subunits are ready to attach to the start codon of another message and start translation anew.
Post-Translational Modifications
Most proteins go through an extensive series of processing steps, collectively called posttranslational modification, before they are ready to go to work in a cell.
Molecular chaperones speed folding of the protein. Folding determines a protein's shape and therefore its function.
What Is the Molecular Basis of Mutation?
A mutation is any permanent change in an organisms DNA.

DNA mutations change the genotype of a cell. This leads to the production of novel types of proteins and so can affect phenotype.
Point Mutations
A single base change is called a point mutation. Point mutations can result from errors in DNA replication.
Unrepaired Mistakes in DNA Synthesis Lead to Point Mutations

Base-pair mismatch Like original DNA Changed DNA replication base pair MUTANT
3 5
Original DNA
5 3
DNA replication
Point Mutations
A point mutation that causes a change in the amino acid sequence of the protein is called a missense mutation. Such mutations are often deleterious, meaning they reduce an individuals fitness.
For example, sickle-cell disease results from a missense mutation in the hemoglobin gene. Mutations that do not change the amino acid sequence of the protein are known as silent mutations. Mutations that change the amino acid to a similar amino acid and therefroe there is not phenotypic change are called neutral mutations.
Sickle-Cell Disease Results from a Point Mutation in the Gene for Hemoglobin
DNA point mutation can lead to a different amino acid sequence. DNA sequence 5 of non-template (coding) strand Amino acid sequence Normal Normal red blood cells DNA sequence 5 of non-template (coding) strand Amino acid sequence Mutant Sickled red blood cells 3 3 Phenotype
Chromosome-Level Mutations
Mutations can also involve larger-scale changes in the composition of chromosomes.

For example, changes in chromosome number can occur -. These result from chance mistakes in the partitioning of chromosomes during meiosis or mitosis.
Chromosome-Level Mutations
The composition of individual chromosomes can also change.

Inversion occurs when a chromosomal segment detaches, flips, and reattaches to the chromosome. Chromosomal translocation occurs when a chromosomal segment detaches and becomes attached to a different chromosome.
Figure 12-6bc
Translocations are abnomalities - translocation

Pieces of chromosomes have been swapped
Leads to uncontrolled cell growth chronic myelogenous leukemia
Sex chromosomes in Klinefelter syndrome polyploidy

Two X chromosomes and one Y chromosome
Causes males that are sterile
Key Concepts
Inside ribosomes, mRNAs are translated to proteins
via intermediary molecules called transfer RNAs. Transfer RNAs carry an amino acid and have a threebase-pair anticodon, which binds to a three-baselong mRNA codon. The amino acid carried by the transfer RNA is then added to the growing protein via formation of a peptide bond.
Mutations are random changes in the DNA that may

or may not produce changes in the phenotype.
Key Concepts
After the enzyme RNA polymerase binds to a
specific site in DNA with the help of other proteins, it catalyzes the production of an RNA molecule. The base sequence of the RNA produced is complementary to the base sequence of the DNA template strand.
Some sections of an RNA are encoded by gene
regions called exons, while others are encoded by gene regions called introns. During RNA processing, introns are removed and the ends of the RNA receive a cap and tail.
Key Concepts
Inside ribosomes, mRNAs are translated to
proteins via intermediary molecules called transfer RNAs. Transfer RNAs carry an amino acid and have a three-base-pair anticodon, which binds to a three-base-long mRNA codon. The amino acid carried by the transfer RNA is then added to the growing protein via formation of a peptide bond.
Mutations are random changes in the DNA that

may or may not produce changes in the phenotype.

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Generating information from genes

Transcription and translation Chapter 16

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

Transcription Is the Synthesis of RNA from a DNA Template

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

RNA Polymerase Structure and Function

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

The 3D Structure of RNA Polymerase Bound to DNA

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

Initiation: How Does Transcription Begin in Bacteria?

Transcription of bacterial genes is initiated at specific sections of DNA called

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

Transcription initiation site?

Promoter (on non-template strand) 35 box

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

Initiation: How Does Transcription Begin in Bacteria?

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

In Bacteria, Sigma Plays a Key Role in Initiating Transcription

HOW TRANSCRIPTION BEGINS

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

In Bacteria, Sigma Plays a Key Role in Initiating Transcription

HOW TRANSCRIPTION BEGINS

RNA exit site

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

Elongation and Termination

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

Transcription Terminates When a Hairpin Forms in the RNA

HOW TRANSCRIPTION ENDS

RNA Hairpin loop

Transcription termination signal

1. RNA polymerase reaches a transcription

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

Transcription Terminates When a Hairpin Forms

HOW TRANSCRIPTION ENDS

2. The RNA hairpin causes the RNA strand

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

Transcription and RNA Processing in Eukaryotes

Eukaryotic transcription shares many fundamental characteristics with prokaryotic transcription.

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

Transcription and RNA Processing in Eukaryotes

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

The Startling Discovery of Eukaryotic Genes

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

Processing of Eukaryote RNA transcripts

Intron 1 Intron 2 DNA 3 5 Exon 1

Primary RNA transcript 5 Spliced transcript 5 3

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

Exons, Introns, and RNA Splicing

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

Introns Are Spliced Out of the Original mRNA

1. Several snRNPs and

2. The 5 end of the

3. The 3 and 5 ends

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

Additional processing - Adding Caps and Tails to RNA Transcripts

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

In Eukaryotes, mRNAs Are Given a Cap and a Tail

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

Copyright 2008 Pearson Education Inc., publishing as Pearson Addison-Wesley

Ribosomes Are the Site of Protein Synthesis