chapter 17 from gene to protein. beadle & tatum bread mold neurospora crassa created mutants...

Chapter 17

From Gene to Protein

Beadle & Tatum• Bread mold • Neurospora crassa• Created mutants using X-rays• That differed from the wild

type in their nutritional needs.

EXPERIMENT:Control: minimal medium- wild

type only- all growClass I: minimal medium +1

additional nutrientClass II: minimal medium +1

additional nutrientClass III: minimal medium +1

additional nutrientThe mediums the mutant could grow in revealed the enzyme pathway and the defective enzyme.

The “one gene - one enzyme” hypothesis states :

• The function of a gene is to dictate the production of a specific enzyme.– Exception: Keratin and insulin are not

enzymes.• Refined to the:• “One gene - one polypeptide”

hypothesis.

The language of DNAis universal.

This means that a gene can be transplanted fromone species to another.

Ex.Firefly gene in a tobaccoplant genome.

Ex.Gene for human insulin in a bacterium.

Question:

How exactly does a gene end up making a protein?

Tell your friend in as much detail as you can in the next 2 minutes. Then switch… let your friend tell you what else they know.

DNA --> RNA --> PROTEIN

THE ANSWER IS CALLED“The central dogma of modern

biology”:

1. DNA is used to construct 3 kinds of RNA.2. 3 kinds of RNA work together to construct protein.

1st TRANSCRIPTION DNA --> RNA

synthesis of RNA under the direction of DNA (rewritten instructions)

3 steps: 1. Initiation2. Elongation3. termination

2nd TRANSLATION RNA --> PROTEIN

synthesis of a polypeptide under the direction of mRNA(change in the language)

3 steps: 1. Initiation2. Elongation3. termination

If DNA & RNA are both made of only 4 kinds of

nucleotides, how can they “code” for PROTEINS, which are made of 20

different kinds of amino acids?

IT’S A TRIPLET CODE!DNA”triplet”corresponds to a

mRNA “codon”corresponds to a

tRNA “anticodon”Each tRNA carries a single

amino acid… this “covers” the 20 aa

How does it work?

X NO 41=4X NO 42=16 YES!!! 43=64

64 combinations of 3 nucleotides!!!! The genetic code is “redundant”.

How are DNA and RNA different?1) Sugar

• Deoxyribose in DNA• Ribose in RNA2) Shape • Double stranded in

DNA• Single stranded in

RNA3) Base • Thymine in DNA• Uracil in RNA

Different types of RNA:1) Messenger RNA (mRNA)• Single strand of RNA• Synthesized complementary

strand to a gene.• Provides the template used for

sequencing amino acids into a polypeptide.

• Triplet group of three adjacent nucleotides on the mRNA, called a codon, codes for one specific amino acid.

THE GENETIC CODE (mRNA CODONS)

If the gene sequenceReads: 3’AAATTTCCCGGG5’

The mRNA Reads:5’UUUAAAGGGCCC3’

Therefore,Amino Acid seq is:___ ___ ___ ___

THE GENETIC CODE (mRNA CODONS)

If the gene sequenceReads: 3’AAATTTCCCGGG5’

The mRNA Reads:5’UUUAAAGGGCCC3’

Therefore,Amino Acid seq is:Phe Lys Gly Pro

2) Transfer RNA (tRNA)• Short RNA molecules (about 80 nucleotides)

used for transporting amino acids to their proper place on the mRNA template.

• Arranged in a hairpin or clover-leaf shape.• The 3’ end of the tRNA (-C-C-A-3’) is the

amino acid attachment site.• Another portion, specified by a triplet

combination of nucleotides, is the anticodon.

• Anticodon of tRNA is complementary to the codon of mRNA during translation.

• Exact base pairing of 3rd nucleotide is not always required- called “wobble” effect.

TRANSFER RNA tRNA

Figure 17.13b The structure of transfer RNA (tRNA)

3) Ribosomal RNA (rRNA)• Building blocks of ribosomes.• Coordinates the activities of mRNA and tRNA

during translation.• Ribosomes have 2 subunits: large & small• Three/Four binding sites:

1) one for the mRNA, 2) one for a tRNA that carries a growing

polypeptide chain (P site), 3) one for a second tRNA that delivers

the next amino acid (A site). 4) ??? *Also (E site) or exit site… the

P site becomes the E site when the ribosome moves down the mRNA

Figure 17.16 Structure of the large ribosomal subunit at the atomic level

TRANSCRIPTION IS BUILDING MOLECULES OF RNABASED ON THE DNA TEMPLATE (GENES).

How is transcription different in

prokaryotic cells and in eukaryotic

cells?

In prokaryotes: 1) The promoter sequence,

segment of DNA that the RNA polymerase attaches to, may be blocked by proteins at its operator.

2) While RNA polymerase transcribes mRNA the mRNA is immediately translated to a polypeptide (without additional processing)

SIMULTANEOUS TRANSCRIPTION & TRANSLATION…

3) Polyribosomes (multiple)

TRANSCRIPTION AND TRANSLATION COUPLED IN BACTERIA

Transcription In Eukaryotes:Initiation; Elongation;

Termination

Transcription In Eukaryotes:Initiation; Elongation;

Termination1) INITIATION• The promoter sequence is

a region before the actual gene. Within it, the T-A-T-A (TATA box) binds (a/or multiple) transcription factor (s).

• NEXT, RNA polymerase binds to this complex. It unzips the DNA into two strands temporarily.

• is synthesis of the mRNA molecule 5’ --> 3’ by RNA polymerase II at the start point (on the 3’ end of the gene).

• Transcription progresses at a rate of 60 nucleotides per second.

2) ELONGATION

3) Termination• occurs when the RNA

polymerase reaches a special sequence of DNA nucleotides that serve as a termination point- terminator.

• The termination sequence is an inverted repeat of GC-rich sequence followed by 4 or more adenosines (AAAAAA)m

• RNA Polymerase is released from the DNA template.

IN EUKARYOTES the mRNA is MODIFIED before leaving the nucleus (not in prokaryotes):

a. The 5’ end: 5’ cap (P-P-P-G-5’) modified guanosine tri-phosphate GTP provides stability to the mRNA and a point of attachment for the ssu of the ribosome

b. The 3’ end: poly-A-tail (150-200 adenosines) provides stability and controls the movement of the mRNA across the nuclear envelope.

c. Splicing (see next slide for details.)

RNA processing: 5’ cap & poly A tail

c) Sections of the primary transcript are removed others are bonded together! RNA splicing

d) The primary mRNA transcript has sections called: introns & exons (unedited, called “heterogenous nuclear RNA”)

e) Introns are… intervening sequences that are noncoding.

f) Exons are… sequences that express a code for a polypeptide.

g) A RNA/protein complex called a snRNP “snurp” (small nuclear ribonucleoprotein)

h) Several snRNP’s and additional proteins create a spliceosome.

The spliceosome deletes out the introns and splices the exons together.

Figure 17.9 RNA processing: RNA splicing

Figure 17.11 Correspondence between exons and protein domains

A problem from the HUMAN GENOMEPROJECT was that manyfewer genes were discoveredthan proteins.

So 1 gene - 1 protein is wrong???

Alternative splicing can result in multipleproteins createdby one gene.

Transcription:initiation, elongation,

termination

TRANSLATION• Occurs… on

ribosomes outside of the nucleus.

• Ribosomes consist of rRNA and proteins.

• The information on mRNA is read off in 3’s (codon).

• Polypeptides (amino acid chains) are assembled.

Translation

Initiation Stage

1) The 5’ end of mRNA (5’cap) attracts the small ribosomal subunit. Attachment.

2) A molecule of tRNA, with the complementary anticodon: UAC hydrogen bonds to the mRNA start codon: AUG.The 1st amino acid on the tRNA is methionine

3) The large sub-unit of the ribosome attaches forming a complete ribosome with the methionine tRNA & mRNA

4) The 1st tRNA is now occupying the P site.

Elongation Stage5) The next codon is read off as

another tRNA (bearing an amino acid) binds to the A site of the ribosome.

6) The amino acids attached to the the P-site and A-site tRNA’s are peptide bonded together.

7) The ribosome translocates (moves) the tRNA in the A site (containing the polypeptide chain) to the P site. The P site tRNA moves to the E site and exits. The A site is now open again for a new tRNA to bring the next AA.

8)The ribosome shifts the mRNA through, one codon at a time… translocation.

Termination Stage9) Translation continues until a ribosome

encounters one of three “stop” codons: UAA, UAG, UGA (cave man talk?)

• Which all bind RELEASE FACTOR instead of an actual tRNA.

• The completed polypeptide, the last tRNA, and the two ribosomal subunits are released.

Translation:initiation,

elongation,termination

WHAT HAPPENS WHEN WE HAVE ERRORS IN THE DNA CODE????

MUTATIONS!!!!

POINT MUTATIONS1) Substitution- switched pair

of nucleotides for the right pair. Can switch the identity of a single amino acid.

• Since most amino acids have multiple codons, they don’t always result in a mutation!!!

• ie: GAU and GAC both code for Asparagine

• GCU, GCC, GCG, and GCA all code for Alanine!

DNA Mutations That Alter Translation:Missense mutation

• base-pair substitution of one nucleotide and its partner for another pair.

• altered codon still codes for an amino acid (still makes sense) but not necessarily the right sense.

• Does not always alter the protein.

ex. Sickle Cell Anemia is caused by a substitution of A for T, therefore Valine instead of Glutamic Acid, in the polypeptide chain alters the shape of hemoglobin.

Nonsense Mutation• Alterations that change

an amino acid codon to a stop signal.

• Translation will be terminated prematurely.

• The polypeptide will be shorter than it should be.

• Always leads to a nonfunctional protein.

2) Insertion/Deletion• Frameshift mutations are

caused by insertions or deletions of nucleotide pairs in a gene.

• since the mRNA is read as a series of nucleotide triplets- insertions or deletions of more than or fewer than 3 nucleotide pairs results in a change of the reading frame.

• This changes the identity of all the amino acids downstream of the insertion/deletion.

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

The processing of genetic information is imperfect and is a source of genetic

variationChanges in genotype can result in changes in phenotype.1. Alterations in a DNA sequence can lead to changes in the type

or amount of the protein produced and the consequent phenotype.

2. DNA mutations can be positive, negative or neutral based on the effect or the lack of effect they have on the resulting nucleic acid or protein and the phenotypes that are conferred by the protein.

3. Errors in DNA replication or DNA repair mechanisms, and external factors, including radiation and reactive chemicals, can cause random changes, e.g., mutations in the DNA.

4. Changes in genotype may affect phenotypes that are subject to natural selection. Genetic changes that enhance survival and reproduction can be selected by environmental conditions.

Evidence:• Whether or not a mutation is detrimental, beneficial or neutral

depends on the environmental context. Mutations are the primary source of genetic variation.

• Errors in mitosis or meiosis can result in changes in phenotype.• Changes in chromosome number often result in new

phenotypes, including sterility caused by triploidy and increased vigor of other polyploids.

• Changes in chromosome number often result in human disorders with developmental limitations, including Trisomy 21 (Down syndrome) and XO (Turner syndrome).

• Antibiotic resistance mutations • Pesticide resistance mutations • Sickle cell disorder and heterozygote advantage• Selection results in evolutionary change. 

Learning Objectives:

• LO 3.24 The student is able to predict how a change in genotype, when expressed as a phenotype, provides a variation that can be subject to natural selection. [See SP 6.4, 7.2]

• LO 3.25 The student can create a visual representation to illustrate how changes in a DNA nucleotide sequence can result in a change in the polypeptide produced. [See SP 1.1]

• LO 3.26 The student is able to explain the connection between genetic variations in organisms and phenotypic variations in populations. [See SP 7.2]