Chapter 17 From Gene to Protein

Lecture Overview

• What is a Gene?

• The Central Dogma

• The Genetic Code

• Transcription

• Translation

• Gene Expression in Eukaryotes and Prokaryotes.

• Mutations

A tobacco plant expressing a firefly gene

What is a Gene?

• Why are dwarf pea plants short?

• An enzyme used to make plant growth hormones

called gibberellins is not made. This is due to an

allele of the enzyme that does not code for a

functional enzyme.

What is a Gene?

• In 1909 Archibald Garrod hypothesized that inherited disease came about because the individual is not able to make a particular enzyme.

• In the 1930’s Beadle and Tatum studied mutants of the bread mold Neurospora. They deduced that a series of mutants that could not make arginine each made a different enzyme in the pathway.

• One gene → one enzyme.

Figure 17.2 Beadle and Tatum’s evidence for the one gene-one enzyme hypothesis

What is a Gene?

• As researchers discovered the roles of proteins that did not code for enzymes, the hypothesis was revised.

• One gene → one polypeptide.

The Central Dogma

Write it down here:

Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information

Figure 17.4 The triplet code

What is the genetic code?

• How do four different chemical bases provide the information for synthesizing all the proteins in an organism?

• A one letter code could only specify four amino acids, yet there are 20.

• A two letter code could specify 4² or 16 different arrangements.

• A three letter code, 4³ = 64 possible arrangements.

• The genetic code is based on codons made up of three nucleotides – the triplet code.

How the Code is Read

• The DNA strand that is transcribed is called the template strand. The complimentary RNA is made from it.
• Each triplet codon on the mRNA corresponds to a particular amino acid. The code is read 5’ to 3’.

Cracking the Genetic Code

• In the 1960s, Marshall Nirenberg synthesized artificial mRNAs and determined the sequence of the peptides synthesized from them.

• A string of U’s always produced phenylalanine, so therefore the codon was UUU.

• Poly A always produced lysine, so therefore the codon for lysine was AAA.

• GGG was the codon for glycine, and CCC was the codon for proline.

Figure 17.5 The dictionary of the genetic code

Features of the Genetic Code

• The genetic code consists of codons of three amino acids.

• Each codon specifies a particular amino acid.

• AUG, which codes for methionine, is the start codon, so methionine is always the first amino acid in the new chain.

• The genetic code is read 5’ to 3’.

• The stop codons, UAG, UGA, & UAA, signal the end of translation of the peptide.

• Reading occurs continuously, without punctuation between the codons, and without overlap.

Since the genetic code is practically universal, it must have evolved
VERY early in life’s history.

Transcription

• RNA Polymerase

• Promoter

• The Transcription Process

• Posttranscriptional Modifications

Transcription is catalyzed by

• Catalyzes the addition of ribonucleotide to the growing 3’ end of an RNA chain.

• No primer is needed

• Synthesis proceeds in the 5’ to 3’ direction.

• Only one of the two DNA strands is transcribed – the ‘template strand’.

• The RNA transcript is single stranded.

• The transcript has the same sequence as the coding strand, except U is used instead of T.

Transcription

• The Promoter: DNA sequences that allow the RNA polymerase to bind to the DNA and start transcription.

• The terminator sequence signals the end of transcription.

• The transcription unit the stretch of DNA that is transcribed to make an RNA molecule.

The Transcription Process

• Initiation

• Elongation

• Termination

Initiation of Transcription (Fig. 17.7)

• Binding of RNA polymerase to the promoter.

• Once bound, the RNA polymerase unwinds the DNA helix to open it up.

Elongation (Fig. 17.7)

• Transcription usually starts with ATP or GTP.

– This nucleotide forms the 5’ end of the chain, which grows 5’ to 3’.

– A primer is not required.

Elongation (Fig. 17.7)

• The open region of DNA containing the RNA polymerase, and growing chain is called the ‘transcription bubble’.

• The RNA polymerase moves down the DNA at about 50 nt/sec

• RNA polymerase has no proofreading capability, errors are not unusual.

Termination of Transcription

• Transcription stop signals are sequences that form hairpin structures, which cause the RNA polymerase to fall off the DNA.

Termination of Transcription

• The RNA strand dissociates from the DNA.

Figure 17.8 The initiation of transcription at a eukaryotic promoter

Posttranscriptional Modifications (Fig. 17.9)

• Required in eukaryotes for exit from the nucleus.

• 5’ cap

– a 5’-5’ linkages with a GTP

– Protects the RNA template from degradation

• 3’ poly-A tails

– 3’ end of the transcript is cleaved off and a poly-A polymerase adds about 250 A ribonucleotides.

The coding sequence of eukaryotic genes is interrupted with segment of non-coding DNA.
(Fig. 17.10)

Exons – coding DNA

Introns – non-coding DNA

• Introns must be removed from transcripts before translation occurs.

How Introns are Removed (Fig. 17.11)

• The first product of transcription is a primary RNA transcript of the entire gene.

• Small catalytic RNAs called snRNPS associate with certain proteins to form a splicesosome .

• The splicesosome folds the primary transcript into loops, and holding the exons close to each other. The introns are cut out.

Figure 17.11 The roles of snRNPs and spliceosomes in mRNA splicing

Why Introns?

1. Alternative splicing creates multiple polypeptides from the same gene.

– Rat calcitonin gene

• Hormone when spliced in parathyroid

• Neurotransmitter when spliced in the brain

Why Introns?

2. Split genes facilitate the evolution of new and potentially useful proteins.

– Proteins are often modular – consisting of several domains. Recombination between genes can create proteins with novel function.

Figure 17.12 Correspondence between exons and protein domains

Translation

• The role of mRNA, tRNA and rRNA.

• Charging tRNA

• “Start” and “Stop” Signals

• Initiation

• Elongation

• Translocation

• Termination

The Role of RNA in Protein Synthesis

• Ribsosomal RNA (rRNA)

– Used to make Ribosomes, the protein synthesis factories.

• Transfer RNA (tRNA)

– Brings the proper amino acid to the ribosome

• Messenger RNA (mRNA)

– Template for protein synthesis.

Table 17.1 Type of RNA

Figure 17.13 Translation: the basic concept

Figure 17.14a The structure of transfer RNA (tRNA)

tRNA

• The RNA strand of the tRNA is folded to make three hairpins with loops.

• At the bottom loop is an ‘anticodon’ which has the complimentary base sequence for the codon corresponding to the amino acid it can carry.

• There is an amino acid attachment site at the 3’ end.

Figure 17.14b The structure of transfer RNA (tRNA)

Activating Enzymes “Read” the Genetic Code

• Aminoacyl-tRNA synthetases attach specific amino acids to the amino acid attachment site of the 3’ end of tRNAs.

• There is a specific aminoacyl-tRNA synthetase for each of the 20 amino acids.

• Each aminoacyl-tRNA synthetase can recognize the corresponding anticodon/s for the amino acid it carries.

Figure 17.15 An aminoacyl-tRNA synthetase joins a specific amino acid to a tRNA

“Start” signals

• Start signal for translation: AUG

• Also codes for the amino acid methionine.

• Ribosome recognizes a ribosomal binding site in the mRNA.

• Usually the first AUG codon after that is the signal of the start of translation.

“Stop” Signals

• There is no tRNA with an anticodon complementary to three of the 64 codons:

– UAA, UAG, and UGA.

• These ‘nonsense’ codons indicate the end of the polypeptide.

Initiation of Translation

• An initiation complex is formed.

– The mRNA binds the small ribsomal subunit.

– tRNA^MET with anticodon UAC base-pairs with the start codon.

– The large ribosomal subunit binds, forming the P, A, and E sites.

• (P = petidyl site, A = aminoacyl site, E = exit site.)

– Initiator proteins facilitate this process.

Figure 17.16 The anatomy of a ribosome

Figure 17.17 The initiation of translation

Elongation

• tRNA^fMET is bound to the P site.

• When a tRNA molecule with the appropriate anticodon appears, elongation factor proteins assist it in bind to the expose mRNA codon at the A site.

• An RNA molecule in the large ribosomal subunit catalyzes the formation a a peptide bond between the two amino acids.

Figure 17.18 The elongation cycle of translation

Translocation

• After the peptide bond is formed between the two amino acids, the ribosome now moves three more nucleotides along the mRNA molecule in the 5’ to 3’ direction.

• The initial tRNA is ejected from the ribosome, the second tRNA is now in the P-site, and the A-site is free.

Figure 17.18 The elongation cycle of translation

Termination

• Elongation continues until a stop codon comes up.

• Release factor proteins recognize the stop codon and release the newly made peptide from the ribosome.

Figure 17.19 The termination of translation

Polyribosomes (Fig. 17.20)

• A single mRNA is used to make many copies of a polypeptide simultaneously.

• A number of ribosomes work on translating the message at the same time.

Targeting of polypeptides to specific locations in the cell.

• Ribosomes in the cytosol are involved in synthesis of soluble proteins, such as enzymes.

• Ribosomes on the rough endoplasmic reticulum RER synthesize proteins that will be bound to the plasma membrane or to the membranes of organelles.

Figure 17.21 The signal mechanism for targeting proteins to the ER

Figure 7.16  Review: relationships among endomembranes

Table 17.1 Types of RNA in a Eukaryotic Cell

Differences between Bacterial and Eukaryotic Gene Expression.

Eukaryotes

• In eukaryotes, transcription occurs in the nucleus, and translation occurs in the cytoplasm, so the two are not linked.

• Most eukaryotic genes posses introns, prokaryotes do not.

• In eukaryotes, mRNA is processed before it leaves the nucleus:

– 5’ cap

– Poly A tail added

– Introns are spliced out

Figure 17.26 A summary of transcription and translation in a eukaryotic cell

Differences between Bacterial and Eukaryotic Gene Expression.

Prokaryotes

• Prokaryotic genes are often organized in operons of genes needed for a certain task, that are all synthesized at once.

• In prokaryotes, transcription and translation of the same gene can occur simultaneously.

Figure 17.22 Coupled transcription and translation in bacteria

Mutations

• A mutation is a change in the DNA nucleotide sequence.

• Point mutations involve a change in just one base pair.

• Substitutions – the replacement with one nucleotide and its pair with another pair of nucleotides, ie. A-T with G-C,T-A, or CG.

Figure 17.23 The molecular basis of sickle-cell disease: a point mutation

Consequences of Base Substitutions

• Change in nucleotide sequence, but the new codon specifies the same amino acid.

• Changes in the nucleotide sequence so that just one or a couple amino acids are different.

– The degree of effect depends on whether the new amino acids have the same properties as the original ones.

– This is called a missense mutation.

• A nonsense mutation is the replacement of an amino acid codon with a stop codon. A truncated non-functional protein is produced.

Figure 17.24 Base-pair substitution

• Additions or losses of one or two base pairs cause a shift in the reading frame of the rest of the protein.

– A frame shift mutation.

• The polypeptide made is usually non-functional.

Figure 17.25 Base-pair insertion or deletion

What is a Gene?

• A region of DNA whose final product is either a polypeptide or an RNA molecule?

The End