Chapter 17~ From Gene to Protein

Protein Synthesis: overview

One gene-one enzyme hypothesis (Beadle and Tatum)

One gene-one polypeptide (protein) hypothesis

Transcription : synthesis of RNA under the direction of DNA (mRNA)

Translation : actual synthesis of a polypeptide under the direction of mRNA

DNA and types of RNA work together to make polypeptides that form proteins in a cell

The Triplet Code

The genetic instructions for a polypeptide chain are ‘written’ in the DNA as a series of 3-nucleotide ‘words’

mRNA = Codons

‘U’ (uracil) replaces ‘T’ in RNA

DNA RNA

2 strands

Thymine

Deoxyribose

Nucleus

Triplet code

One type

1 strand

Uracil

Ribose

Nucleus/cytoplasm

Codon/Anticodon

3 types mRNA, tRNA, rRNA

Transcription & Translation

They are the two main steps form a gene to a protein.

A gene is a segment of DNA that codes for a protein product.

RNA links DNA’s genetic instructions for making proteins.

Transcription: Making RNA from DNA

Translation: ‘translating’ the RNA into a protein

Remember that RNA is different from DNA in that:

It is single stranded.

Has ribose instead of deoxyribose

Has Uracil instead of thymine

The linear sequence of nucleotides (A, C, G, T) in DNA ultimately determines the linear sequence of amino acids in a protein (the primary structure).

Genes can be hundreds or thousands of nucleotides long.

Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information

Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information

Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information

Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information

Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information

Figure 17.3 The triplet code

Transcription:

Genes are transcribed from DNA into mRNA.

DNA is read as triplets (codons).

Codon: is 3 nucleotides long and codes for a specific amino acid.

Codons are the “words”; when the amino acids they make are linked together, they make the sentence (protein).

Genes are not directly translated into amino acids, but first are transcribed as codons in mRNA.

Figure 17.3 The triplet code

Transcription, I

RNA polymerase : pries DNA apart and hooks RNA nucleotides together from the DNA code

Promoter region on DNA : where RNA polymerase attaches and where initiation of RNA begins

Terminator region : sequence that signals the end of transcription

Transcription unit : stretch of DNA transcribed into an RNA molecule

Transcription, II

Initiation ~ transcription factors mediate the binding of RNA polymerase to an initiation sequence (TATA box)

Elongation ~ RNA polymerase continues unwinding DNA and adding nucleotides to the 3’ end

Termination ~ RNA polymerase reaches terminator sequence

Transcription: overview

Transcription:

DNA is a double helix, however, only 1 of these strands is transcribed.

The strand that is transcribed is called the template strand (sense strand).

The other strand is NOT transcribed and is called the non-template strand (nonsense strand); it serves as a template for making a new strand during replication.

Transcription:

An mRNA will be complementary to the DNA template from which it is transcribed.

Ex: if DNA reads “CCG” then the RNA that is made will read “GGC”

Try: DNA reads “CAT”

Then: RNA reads “GUA”

Remember that RNA uses uracil instead of thymine.

Similar to DNA replication (RNA primer), there is a promoter region that is required to start the production of mRNA

RNA pol will bind to this promoter region (~ 100 nucleotides long).

RNA pol can’t recognize the region without the help of transcription factors.

Transcription factor:

TATA box: A short nucleotide sequence at the promoter region that is T and A rich.

The box is ~ 25 nucleotides ‘upstream’ from the initiation site.

Once transcription begins, RNA pol II moves along DNA and performs 2 functions:

Functions of RNA pol II

1. It untwists & opens a short segment of DNA exposing about nucleotides.

2. It links incoming RNA nucleotides to the 3’ end of the elongating strand. Therefore, RNA grows one nucleotide at a time in the 5’ à 3’ direction.

Figure 17.7 The initiation of transcription at a eukaryotic promoter

As the mRNA strand elongates, it peels away from the DNA template.

It can grow ~ 30-60 nucleotides per second.

Transcription continues until it reaches a termination sequence.

In eukaryotes, the most common sequence is ATAAA.

Figure 17.6 The stages of transcription: initiation, elongation, and termination

Figure 17.6 The stages of transcription: initiation, elongation, and termination

Figure 17.6 The stages of transcription: initiation, elongation, and termination

Figure 17.6 The stages of transcription: initiation, elongation, and termination

Translation

The synthesis of a polypeptide, which occurs under the direction of mRNA.

The mRNA made in transcription leaves the nucleus and then travels into the cytoplasm to be translated.

Translation

Translation occurs on the ribosomes.

Ribosomes are made of rRNA

Ribosomes facilitate the orderly linking of amino acids into polypeptide chains.

Figure 17.0 Ribosome

mRNA modification

1) 5’ cap: modified guanine; protection; recognition site for ribosomes

2) 3’ tail: poly(A) tail (adenine); protection; recognition; transport

3) RNA splicing: exons (expressed sequences) kept,introns (intervening sequences) spliced out; spliceosome

Translation, I

mRNA from nucleus is ‘read’ along its codons by tRNA’s anticodons at the ribosome

tRNA anticodon (nucleotide triplet); amino acid

Translation, II

rRNA site of mRNA codon & tRNA anticodon coupling

P site holds the tRNA carrying the growing polypeptide chain

A site holds the tRNA carrying the next amino acid to be added to the chain

E site discharged tRNA’s

Translation, III

Initiation ~ union of mRNA, tRNA, small ribosomal subunit; followed by large subunit

Elongation ~ •codon recognition •peptide bond formation •translocation

Termination ~ ‘stop’ codon reaches ‘A’ site

Translation

Mutations: genetic material changes in a cell

Mutagens: physical and chemical agents that change DNA

Point mutations….

Changes in 1 or a few base pairs in a single gene

Base-pair substitutions: •silent mutations no effect on protein

•missense ∆ to a different amino acid (different protein )

•nonsense ∆ to a stop codon and a nonfunctional protein

Frameshift

Sickle Cell Hemoglobin

During translation, the linear sequence of codons along mRNA is translated into the linear sequence of amino acids in a polypeptide.

Because codons are triplets, the # of nucleotides making up a polypeptide is 3 times the # of amino acids.

By the 1960’s, all 64 codons were decoded.

61 out of 64 code for amino acids.

The triplet AUG has 2 f(x)’s: start and Methionine.

Three codons code for signal termination (stop): UAA, UAG, & UGA

There is redundancy in the genetic code, but no ambiguity.

Redundancy exists because 2 or more codons can code for the same AA.

Ex: UUU & UUC both code for the amino acid Phenylalanine (Phe)

There is no ambiguity; each codon can only code for one type of amino acid.

OR: each codon can only do 1 specific thing

Reading Frame:

The correct grouping of nucleotides is important in the molecular language.

Sequences of amino acids are only produced if the correct codons are grouped.

Changes in the reading frame could have no effect or drastic effects.

THE CAT ATE THE RAT.

Adding one letter could change the meaning.

THE CAT SAT ETH ERA T.

The genetic code is universal:

Among every living organism, each codon means the same thing.

Ex: UUG codes for Trp in humans, slugs, flies, fish etc.

** There are some minor exception in ciliates (group of Protists)

Translation:

For translation to occur, there must be 3 things present at the same location:

1. mRNA (has the DNA code)

2. rRNA (the ribosome)

3. tRNA (transfers each amino acid to a growing change)

Figure 17.12 Translation: the basic concept

tRNA

It aligns the appropriate AA’s to form a new polypeptide.

It contains anticodons which have the opposite sequence of the mRNA codon; therefore, they bind.

Each tRNA has the correct AA for each anticodon.

Figure 17.13a The structure of transfer RNA (tRNA)

Figure 17.13b The structure of transfer RNA (tRNA)

Ex of anticodons & tRNA

The mRNA codon UUU is translated as the AA phenylalanine (Phe).

The tRNA that transfers Phe to the growing chain has an anticodon of AAA.

The ability of tRNA to carry the specific AA for its anticodon depends on its structure.

tRNA

Is only about 80 nucleotides long.

A loop protrudes at one end where the anticodon is located.

At the other end (3’ end) is the attachment site for the correct AA.

Figure 17.15 The anatomy of a functioning ribosome

Figure 17.17 The initiation of translation

Figure 17.18 The elongation cycle of translation

Figure 17.19 The termination of translation

Figure 17.20 Polyribosomes

Figure 17.25 A summary of transcription and translation in a eukaryotic cell

Introns & Exons

Exon = codes for proteins

Introns = no protein product; “junk” DNA

Eukaryotes have more introns that exons.

Why?

Introns:

May control gene activity.

Play a role in the evolution of protein diversity; they increase the probability that recombination (crossing over) will occur between alleles.

Figure 17.9 RNA processing: RNA splicing

Figure 17.10 The roles of snRNPs and spliceosomes in mRNA splicing

Figure 17.11 Correspondence between exons and protein domains

MUTATIONS

Any permanent change in DNA that can involve large chromosomal regions, or a single nucleotide pair.

Mutations:

Point Mutation: limited to one nucleotide in a single gene pair.

1. Base Pair Substitution

2. Insertions or Deletions

Point Mutation: Base Substitution

The replacement of one base pair with another.

Occurs when a nucleotide and its partner from the complementary DNA strand are replaced with another pair of nucleotides.

Base Pair Sub:

Depending on how base-pair substitutions are translated, they can result in little or no change in the protein encoded by the mutated gene.

Redundancy in the genetic code is why some substitution mutations have no net effect.

A base pair change may simply transform one codon into another that codes for the same AA.

The substitution may occur in a intron.

Some mutations can have drastic effects.

Remember sickle cell anemia ?

One AA is changed due to one nucleotide being changed.

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

Progeria

Hutchinson-Gilford progeria is caused by a tiny, point mutation in a single gene, known as lamin A (LMNA).

The genetic mutation appears in nearly all instances to occur in the sperm prior to conception

Nearly all cases are found to arise from the substitution of just one base pair among the approximately 25,000 DNA base pairs that make up the LMNA gene.

Appears on chromosome 8.

Only ~ 100 cases recorded.

The LMNA gene codes for two proteins, lamin A and lamin C, that are known to play a key role in stabilizing the inner membrane of the cell's nucleus.

The mutation responsible for Hutchinson-Gilford progeria causes the LMNA gene to produce an abnormal form of the lamin A protein. The abnormal protein destabilizes the cell's nuclear membrane in a way that may be particularly harmful to tissues routinely subjected to intense physical force.

Interestingly, different mutations in the same LMNA gene have been shown to be responsible for at least a half-dozen other genetic disorders, including two rare forms of muscular dystrophy.

Base Pair Sub:

Mis-sense mutation: substitution that alters an AA codon

Non-sense mutation: a substitution that goes from coding an AA into a stop codon.

Point Mutations: Insertions/Deletions

Usually have a greater negative effect on proteins than substitutions.

Inserting or deleting one single nucleotide will alter the reading frame.

This is called a frameshift mutation.

This most likely will produce a non-f(x)’al protein.