13 Gene Expression

Lecture Outline

I. Most genes carry information for making proteins

A. In 1908, Archibald Garrod proposed that genes are responsible for enzymes while studying a rare genetic disease, alkaptonuria

1. Garrod published a book: Inborn Errors of Metabolism

2. Sumner, in 1926, purified a different enzyme and found it to be a protein (Nobel Prize in 1946)

B. In the 1940s, George Beadle and Edward Tatum worked on Neurospora (a mold)

1. 1 mutant gene affected 1 single step in a metabolic pathway

2. The A one gene, one enzyme@ hypothesis

a) It= s now known that some genes encode proteins that are not enzymes; based on the work of Pauling and others

b) It= s also known that one gene is responsible for one polypeptide chain, and that some proteins have more than one chain

c) Beadle and Tatum used the term A wild type,@ meaning the organism with the normal phenotype; it does not imply dominant or recessive, just the normal

3. The A one gene, one polypeptide@ hypothesis

II. DNA is transcribed to form RNA; RNA is translated to form a polypeptide

A. RNA (ribonucleic acid) is used as an intermediary between DNA and proteins

1. RNA is a single-stranded, uncoiled nucleotide polymer (although some areas may be folded)

2. The sugar in RNA is ribose, and uracil substitutes for thymine

a) Uracil is a pyrimidine and forms two bonds with adenine

B. A copy of the DNA is made in the form of messenger RNA (mRNA) in transcription

C. Translation involves mRNA, tRNA (transfer RNA), and rRNA (ribosomal RNA) coordinating to produce proteins

1. mRNA has sequences of 3 nucleotides called codons

a) Codons are read in sequences of 3; this is called the triplet code

b) Codons are written in a 5' to 3' fashion

c) Each codon codes for 1 amino acid

d) 4 bases can combine in 43 combinationsC more than enough to code for the 20 naturally occurring amino acids

2. Each tRNA molecule has a sequence of 3 nucleotidesC the anticodon

a) Anticodons base pair with the codon in a complimentary way via hydrogen bonds

b) Anticodons are written in a 3' to 5' fashion

3. Ribosomes are composed of proteins and rRNA

D. Biologists cracked the genetic code in the 1960s

E. In 1961, Crick and colleagues determined that the code was read in a nonoverlapping sequence of bases, forming a 3-base reading frame

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1. 1 nucleotide code for 1 amino acid? No, 4 bases, 20 amino acids

2. 2 nucleotides code for 1 amino acid? No, not enough combinations

3. 3 nucleotides code for 1 amino acids? Yes, 64 possible combinations

F. Sidney Brenner, in 1961, concluded from experiments that it was a nonoverlapping triplet code; he established the A reading frame@

G. Nirenberg and Matthaei produced experimental evidence that began assigning mRNA to base sequences

1. First was poly-U (phenylalanine = UUU)

2. In 1968, Nirenberg received the Nobel Prize

3. Further found that UAA, UGA, and UAG did not code for any amino acid; they are stop codons

4. By 1967, the code was completely A cracked@

5. Remember that the code is in mRNA code

6. The genetic code of all 64 possible codons has been found to be nearly universal

7. The genetic code is redundant

a) Redundant codons have similar base sequences

b) Only methionine and tryptophan have single triplet codes

H. The wobble hypothesis, proposed by Crick, suggests that the third nucleotide of the tRNA anticodon can bind with more than one nucleotide of the mRNA codon, as cells typically produce only about 40 different tRNA molecules

III. Transcription is the synthesis of RNA from a DNA template

A. RNA is synthesized by DNA-dependent RNA polymerases, which are similar to DNA polymerases

B. Like DNA replication, the reactions are exergonic and require an input of ATP

C. Messenger RNA contains base sequences that code for protein

1. Transcription does not involve a primer; it begins at a promoter sequence on the template strand (the promoter is not transcribed)

2. RNA synthesis proceeds in a 5' ± 3' direction (copying the DNA from the 3' ± 5')

a) Upstream refers to sections toward the 5' end of the mRNA sequence

b) Downstream refers to sections toward the 3' end of the mRNA sequence

D. Synthesis of mRNA includes initiation, elongation, and termination

1. In both prokaryotes and eukaryotes, RTNA polymerases move past the promoter to begin transcription of DNA

2. Bacterial promoters are about 40 bases long and are located in the DNA, just upstream from the starting point

3. Unlike DNA synthesis, RNA synthesis does not require a primer

4. In elongation, nucleotides are added in a complementary way with the input of energy

5. Elongation continues until RNA polymerases recognize a stop signal

6. At the stop signal, the process differs in prokaryotes and eukaryotes

a) In prokaryotes, transcription stops immediately

b) In eukaryotes, RNA polymerase continues to add new nucleotides for about 10B 15 nucleotides past the stop

 

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7. Typically only one strand of the DNA is transcribedC the template strand

a) In a single DNA molecule, some of the template sections are on one strand, others are on the other strand

E. Messenger RNA contains base sequences that do not directly code for protein

1. mRNA has a noncoding leader sequence at the 5' end, important in binding to the ribosome during translation

2. The start codon begins the coding sequences that follow the leader sequence

3. Coding sequences are followed by a stop codon (UAA, UAG, and UGA), and may be followed by variable noncoding 3' trailing sequences

IV. During translation, the nucleic acid message is decoded

A. An amino acid is attached to tRNA before becoming incorporated into a polypeptide

1. To form a polypeptide chain, the amino and carboxyl groups of amino acids are joined by peptide bonds

2. The specific sequence of the amino acids (primary structure) is dictated by the sequence of codons of the mRNA

3. tRNA molecules are produced from tRNA genes

a) tRNA is linked to amino acids by aminoacyl-tRNA synthetases, forming aminoacyl-tRNAs

b) This is an energy-requiring process

B. tRNA molecules have specialized regions with specific functions

1. tRNA molecules have attachment sites for amino acids

2. tRNA molecules have anticodons that will bind to complementary codons of the mRNA

3. tRNA molecules must be recognized by both the specific aminoacyl-tRNA synthetase and the ribosome and have recognition sites for each

4. tRNA molecules are approximately 70 nucleotides long, with some generic sections and some unique sections

5. The nucleotide chain is folded back upon itself to form 3 or more loops, with unpaired nucleotides exposed to form the recognition or attachment sites

C. The components of the translational machinery come together at the ribosomes

1. Ribosomes are composed of two subunits

a) In E. coli, the small subunit is composed of 21 proteins and 1 RNA molecule; the large subunit is composed of 35 proteins and 2 RNA molecules

b) The large subunit has a groove into which the small subunit fits

c) Ribosomes are transcribed from DNA but do not carry informationC rather, they function as the physical site of translation and as a catalyst

d) The A (aminoacyl) site of the ribosome is where the aminoacyl-tRNA binds

e) The P (peptidyl) site of the ribosome is where the tRNA holding the polypeptide chain is positioned

f) The E (exit) site is where the tRNAs that have delivered the amino acids to the growing polypeptide chain exit the ribosome

D. Translation includes initiation, elongation, and termination

1. Initiation is the first step

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a) Initiation factors (proteins) move an initiation tRNA onto the small ribosomal subunit

b) The codon for the initiation is AUG, which codes for the amino acid methionine (met), although the methionine is often removed later

c) The initiation complex binds to ribosome-recognition sequences on the mRNA and aligns the anticodon of the tRNA with the codon of the mRNA

d) The large ribosomal subunit then binds, forming the functional ribosome or initiation complex

e) In eukaryotes, the methionine of the initiator tRNA is unmodified; there is no leader sequence, but a start codon is embedded in a short sequence; and lastly, the initiation complex is more complex

2. Elongation is the addition of new amino acids to the growing protein

a) As the initiator, tRNA is bound to the P site of the ribosome; the A site is unoccupied until the next aminoacyl-tRNA moves in

b) The energy for this binding process comes from guanosine triphosphate(GTP)

c) Peptide bond formation takes place between the amino group of the new amino acid and the carboxyl group of the amino acid bound in the preceding reaction

d) The tRNA molecule is released from the P site

(1) This reaction requires the ribozyme, peptidyl transferase

(2) This ribozyme is not a protein but is an rRNA portion of the large ribosomal subunit

e) Protein synthesis always proceeds from the amino end to the carboxyl end

f) Translocation is the movement of the growing polypeptide chain from the A site to the P site

(1) Energy for translocation comes from GTP

g) The tRNA, without an attached amino acid, now moves from the P to the E site

h) This tRNA then leaves the ribosome and becomes part of the tRNA pool in the cytosol

i) Translation of the mRNA proceeds from a 3' to a 5' direction, which is the same as the direction of transcription

j) Termination occurs when the mRNA presents the codons UAA, UGA, or UAG, as there is no tRNA that is complementary to them

(1) Release factors recognize these codons, and a bond between the tRNA in the P site and its amino acid breaks

(2) The ribosome dissociates into the 2 subunits, and the newly produced polypeptide chain dissociates from the ribosome

E. A polyribosome is a complex of one mRNA and many ribosomes

1. In prokaryotes, transcription and translation occur simultaneously

2. The half-life of the mRNA molecules in bacteria is about 2 minutes

V. Transcription and translation are more complex in eukaryotes than in prokaryotes

A. Eukaryotic mRNA molecules are processed after transcription; prokaryotic mRNAs are used immediately without processing

1. Processing begins in the nucleus; 5' cap is added to the 5' end

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a) The cap is 7-methylguanylate, which is GMP and a methyl group added

b) The cap is necessary for binding to ribosomes

2. Capping may protect mRNA from degradation; half-lives of eukaryotic mRNA molecules are as long as 24 hours

3. At the 3' end of the molecule, a sequence is added that acts as a signal for the subsequent addition of a tail

4. Enzymes recognize the signal and add 100B 250 adenines (the poly-A tail)

B. Both noncoding sequences and coding sequences are transcribed from eukaryotic genes

1. Genes of most eukaryotes have interrupted coding sequences

a) Coding sequences are exons (expressed sequences); noncoding sequences are introns (intervening sequences)

b) A typical eukaryotic gene has multiple exons and introns, with variable numbers of each

2. This terminology refers to sequences in both DNA and mRNA

3. Precursor mRNA (pre-mRNA) contains both introns and exons

4. Small nuclear ribonucleoprotein complexes (snRNPs) may bind to the introns and catalyze excision and splicing

a) To be functional, it must be capped and have a poly-a tail added

5. RNA may also act as a self-acting enzyme and excise and splice without the intervention of protein enzymes

C. The evolution of eukaryotic gene structure is not completely understood

1. In the 1980s, Gilbert proposed that exons are nucleotides that code for protein domains

2. Protein domains are regions of protein tertiary structure with specific functions

3. New proteins with new functions emerge rapidly when genetic recombination produces new combinations of exons and therefore novel proteins

4. This may lead to evolution by exon shuffling

5. One hypothesis states that introns may have evolved from transposons

D. The definition of a gene has changed

1. A gene is a nucleotide sequence which produces a specific RNA or a polypeptide

E. There are exceptions to the usual direction of flow of genetic information

1. In 1964, Temin discovered that some viruses use reverse transcriptase (an RNA directed DNA polymerase) to transcribe DNA from an RNA template

2. Viruses, called retroviruses, use reverse transcriptase

3. HIV-1, which causes AIDS, is the best known retrovirus

4. Reverse transcriptase is a very important research tool for molecular biologists

VI. Mutations are changes in the nucleotide sequence of DNA

A. Base-substitution mutations involve the exchange of one base for another

1. Due to redundancy of the code, this may not alter the product

2. Missense mutations result in the substitution of 1 amino acid for another

a) The resulting protein may be nonfunctional, or the mutation may have no effect on the organism (a silent mutation)

3. Nonsense mutations result in a stop codon

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B. Frameshift mutations involve insertion or deletion of a base pair

1. Frameshift mutations result in an entirely different sequence of amino acids (they change the reading frame)

2. These usually result in a nonfunctional protein

C. Transposons, or transposable genetic elements, are movable sequences of DNA that may move into another area of DNA

1. Transposons may disrupt genes but may also activate some genes

2. Transposons were discovered by Barbara McClintock, in corn, in the 1950s (she received the Nobel Prize in 1983)

3. Transposons have some similarities to retroviruses; most are retrotransposons

D. Hot spots are regions of DNA more likely to undergo mutations and often share regions of repeated nucleotides, causing the polymerases to slip during DNA replication

E. Mutagens are agents that cause mutations, including various types of radiation and chemicals

1. Mutations in somatic cells are not passed on to the next generation

2. Some somatic mutagens are also carcinogens

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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