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Chapter 14

Gene Structure and Expression

What do we need genes for?

Genes contain instruction s for making proteins

14.1 The Connection between DNA, RNA, and Protein


a. Genes specify either protein or RNA products

b. The pathway from gene to polypeptide

involves transcription and translation


c. Genetic code is written in three-letter words

using a four-letter alphabet

How do we know that genes encode proteins?


1. (1896): Garrod studied alkaptonuria disease
- metabolic disorder (proteins and enzymes)
- produce chemical that turns black in air - inherited

alteration in a gene that encodes the enzyme that metabolizes this chemical

How do we know that genes encode proteins?


2. (1940s): Beadle and Tatum studied orange bread mould (Neurospora crassa)
Mould grows on mininal media Used X-rays to produce nutritional mutants (auxotrophs) Hypothesis: Each mutant had defective gene for enzyme needed to synthesize a particular nutrient

Beadle and Tatum Experiment


Arginine Synthesis Pathway

Each step is controlled by a gene that encodes an enzyme for that step

Fig. 14-2, p. 298

Beadle and Tatum Experiment

Fig. 14-2, p. 298

Relationship between genes and proteins


One geneone enzyme hypothesis Direct relationship between genes and enzymes One geneone polypeptide hypothesis Not all proteins are enzymes Functional proteins sometimes contain one or more polypeptides Different genes encode each polypeptide

How to get from genes to proteins?


1. Transcription
Nucleotide sequence in DNA is copied into a complementary sequence in an RNA molecule Template Strand of DNA is used to create messenger RNA (mRNA)

2. Translation
Sequence of nucleotides in mRNA molecule specifies amino acid sequence in polypeptide Ribosome assembles the amino acid sequence

Transcription and Translation

Fig. 14-3, p. 299

Genetic Code
Information
4 nucleotide bases in DNA or RNA 20 different amino acids in polypeptides

Code
One-letter words --> Only 4 combinations Two-letter words --> Only 16 combinations Three-letter words --> 64 combinations

Genetic Code
DNA
Three-letter code: Triplet

RNA
Three-letter code: Codon

complimentary

one codon encodes one amino acid

Genetic Code

Read 35

Read 5-3
U substituted for T Read N terminus C- terminus

Fig. 14-4, p. 299

Features of the Genetic Code


Sense codons 61 codons specify amino acids Most amino acids specified by several codons (_redundancy) Ex: CCU, CCC, CCA, CCG all specify proline

Nucleic acid codes are sequential No commas or spaces between codons Start codon AUG establishes the reading frame

Features of the Genetic Code


Start codon (initiator codon) AUG First codon recognized during translation Specifies amino acid methionine_

Stop codons (termination codons)


End of a polypeptide-encoding mRNA sequence UAA, UAG, UGA

Codons

Fig. 14-5, p. 300

Genetic Code is Universal


Same codons specify the same amino acids in all living organisms and viruses

Genetic code was established very early in the evolution of life and has remained unchanged

14.2 Transcription: DNA-Directed RNA Synthesis


a. RNA polymerases work like DNA

polymerases but require no primer


b. Specific sequences of nucleotides in the DNA

indicate where transcription of a gene begins and ends

Transcription
Info in DNA is transferred to a complementary RNA copy

Similar to DNA replication, except:


Only one DNA strand used as a template Only transcribes the genes RNA polymerases_used RNA are single strands

Uracil replaces thymine

RNA Polymerases
No primers needed to start complementary

copy
RNA is made in the 53direction DNA template strand is read 35

Transcription Overview
Begins as RNA polymerase binds to DNA DNA double helix begins to unwind RNA polymerase adds RNA nucleotides sequentially

according to the DNA template


Enzyme and completed RNA transcript release from

DNA template

Transcription Overview

Transcription Overview

Organization of a Gene
Promoter Control sequence initiates transcription Upstream of transcritional unit Where RNA polymerase binds _Transcription unit_ Portion of gene that is copied into RNA Terminator Signals the end of transcription of a gene

Transcription: Initiation, Elongation, and Termination

Transcription: Initiation, Elongation, and Termination

Initiation
RNA polymerase II Euk. have TATA box in promoter Transcription factors_ bind promoter _RNA polymerase II__binds transcription

factors
Unwind DNA and begin transcription

Termination
Termination of transcription differs in

eukaryotes- polyadenylation signal


Prokaryotes have terminators

14.3 Production of mRNAs in Eukaryotes


a. Eukaryotic protein-coding genes are

transcribed into precursor mRNAs that are modified in the nucleus


b. Introns are removed during pre-mRNA

processing to produce translatable mRNA


c. Introns contribute to protein variability

Messenger RNA
Prokaryotes

Coding region flanked by 5 and 3 untranslated regions (UTRs)


Eukaryotes

Coding region flanked by 5 and 3 untranslated regions (UTRs) Additional noncoding elements (Introns_) found in pre mRNA only, not mRNA

Pre-mRNA
Precursor-mRNA (pre-mRNA) Must be processed in nucleus to produce translatable mRNA 5 cap Reversed _guanine_-containing nucleotide Site where ribosome_ attaches to mRNA Poly(A) tail 50 to 250 adenine_ nucleotides added to 3 end by poly A polymerase Protects mRNA from RNA-digesting enzymes

Pre-mRNA
Introns Non-protein-coding sequences in the premRNA Must be removed before translation

Exons Amino acid coding sequences in pre-mRNA Joined together sequentially in final mRNA

How do we remove introns?

Protects mRNA mRNA splicing

Poly (A) polymerase

Fig. 14-8, p. 305

mRNA Splicing
Introns in pre-mRNAs removed

Spliceosome Pre-mRNA Small ribonucleoprotein particles (snRNP)


Small nuclear RNA (snRNA) + several proteins

mRNA Splicing (contd)


snRNPs

Bind to introns _Loop introns out of the pre-mRNA Clip the intron at each exon boundary

Join adjacent exons together

mRNA Splicing

RNA part does the cutting ribozymes

Fig. 14-9, p. 306

Why are Introns Present?


Alternative splicing _Different versions of mRNA can be produced Exon shuffling Generates new proteins

Protein Variability = Diversity

Alternative Splicing
Exons joined in different combinations to

produce different mRNAs from the same gene


Different mRNA versions translated into

different proteins with different functions


More information can be stored in the DNA

Efficiency

Alternative mRNA Splicing


-tropomyosin in smooth and striated muscle

Similar protein but _different structure _different_ function Fig. 14-10, p. 307

Exon Shuffling
Intron-exon junctions often occur between

major functional regions in encoded protein


Exon Shuffling mixes protein regions or

domains into novel combinations


Allows quicker and more efficient evolution of new proteins

14.4 Translation: mRNA-Directed Polypeptide Synthesis


a. tRNAs are small RNAs of a highly distinctive

structure that bring amino acids to the ribosome


b. Ribosomes are rRNA-protein complexes that work

as automated protein assembly machines


c. Translation initiation brings the ribosomal subunits,

an mRNA, and the first aminoacyl-tRNA together


d. Polypeptide chains grow during the elongation

stage of translation
e. Termination releases a completed polypeptide from

14.4 Translation: mRNA-Directed Polypeptide Synthesis (contd)


d.

Multiple ribosomes simultaneously translate a single mRNA Newly synthesized polypeptides are processed and folded into finished form Finished proteins contain sorting signals that direct them to cellular locations

e.

f.

g.

Base-pair mutations can affect protein structure and function

Translation Overview
Assembly of amino acids into polypeptides

Occurs on ribosomes
P, A, and E sites on ribosome used for

stepwise addition of amino acids to polypeptide as directed by mRNA

Translation Overview

tRNAs
Transfer RNAs (tRNA) Bring specific amino acids to ribosome Cloverleaf shape Bottom end of tRNA contains anticodon

sequence that pairs with codon in mRNAs


Top end contains amino acid

tRNA Structure

Read 3 5
Fig. 14-12, p. 309

Why we have 61 tRNAs?


Wobble Hypothesis
61 different sense codons do not require 61 different

tRNAs
First two nucleotides of anticodon and codon must match exactly Third nucleotide has more flexibility

Ex: tRNA carrying glutamine Matches codons CAA and CAG

Aminoacylation
Adds amino acid to tRNA Aminoacyl-tRNA (amino acid linked to tRNA)

Aminoacyl-tRNA synthetases catalyze reaction

Need ATP to do this.

Ribosomes

Free or Bound

Made of ribosomal RNA (rRNA) and proteins Two subunits: Large and small

Fig. 14-13, p. 310

Translation Stages
1. Initiation Ribosome assembled with mRNA molecule and initiator methionine- tRNA

2. Elongation Amino acids linked to tRNAs added one at a time to growing polypeptide chain 3. Termination New polypeptide released from ribosome Ribosomal subunits separate from mRNA

Initiation
1.
2.

Initiator tRNA (Met-tRNA) binds to small subunit


Complex binds to 5 capof mRNA, scans along mRNA to find AUG start codon Large ribosomal subunit binds to complete initiation

3.

Met-tRNA is in the P site


In prokaryotes there is no 5cap so bind directly to site just before AUG

Initiation
Only one to go directly to P site

Fig. 14-14, p. 311

Initiation
Establishes reading frame

Energy is consumed

Fig. 14-14, p. 311

Elongation

Aminoacyl-tRNA matching the next codon

enters A site
Peptidyl Tranferase catalyzes formation of

first peptide bond and cleaves tRNA in P site - A site is now empty again.

Elongation (contd)
Ribosome moves along mRNA to next codon Empty tRNA moves from _______, then released Newly formed peptidyl-tRNA moves from ___________ A site empty again

Elongation
energy

energy
Fig. 14-15, p. 312

Termination
Begins when A site reaches stop codon Release factor (RF) or termination factor binds

to A site
Polypeptide chain released from P site Remaining parts of complex separated

Fig. 14-16, p. 314

Polysomes
Multiple ribosomes can simultaneously

translate a single mRNA

Fig. 14-17, p. 314

Simultaneous Transcription and Translation


Can occur in prokaryotes (no nuclear envelope)

Fig. 14-18, p. 314

Polypeptide Processing
Processing reactions convert polypeptides into

finished form
Removal of one or more amino acids from the protein chains Addition of organic groups Folding guided by chaperones Alternative pathways to different mature polypeptides

What about proteins in the secretory pathway?


Proteins are distributed within cells by sorting

signals
Signals are coded in DNA, appear when

protein is made

Sorting Signals in ER
Proteins sorted at rough endoplasmic

reticulum
Signal peptide (signal sequence) At beginning of polypeptide chain Signal recognition particle (SRP) Binds to signal peptide -stops translation

Sorting Signals in ER (contd)


SRP receptor SRP binds to protein receptor in ER membrane Ribosome bound onto ER membrane Growing polypeptide pushed inside ER lumen Signal peptidase Removes signal sequence Translation continues until polypeptide complete

Signal Mechanism in ER
Where does it go now? ER, golgi lysosomes, plasma membrane, secreted.

Fig. 14-19, p. 315

Mutations
Changes in genetic material Base-pair mutations change in DNA triplet

Results in change in mRNA codon_


May lead to changes in the amino acid

sequence of the encoded polypeptide

Mutations
1. Misense mutation: Changes a sense codon to

different sense codon


2. Nonsense mutation: Changes a sense codon to a

stop codon
3. Silent mutation: Changes one sense codon to

another sense codon that specifies the same amino acid


4. Frameshift mutation: Base-pair insertion or

deletion alters the reading frame after the point of the mutation

Mutations

Fig. 14-20, p. 316

Mutations

Fig. 14-20, p. 316

Mutations

Fig. 14-20, p. 316

Mutations

Fig. 14-20, p. 316

Mutations

Sickle-Cell Disease
Caused by a single missense mutation

Fig. 14-21, p. 317

Putting it into perspective


1. What is the connection between DNA, RNA and

protein?
2. What is the process of transcription?
3. What is the process of mRNA processing? 4. What is the process of translation?

5. What can happen when there are mutations?

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