General Biology I
Chapter 12 Gene Expression at the Molecular Level
Inborn Errors of Metabolism
In the early 1900s Gregor Mendel’s work on genetics was rediscovered and finally understood.
In 1908 Archibald Garrod, a British Physician who was studying the inheritance of
the disease Alkaptonuria, proposed a link between genes and the production of enzymes.
By 1908 biochemists understood that metabolic pathways consisted of a series of enzymes,
each one converting one chemical into another.
Garrod applied Mendel’s theory on genes to the pathway for the breakdown of
Phenylalanine and the disease Alkaptonuria.
Garrod knew Alkaptonuria was inherited and proposed the disease was caused when
an individual inherited recessive, defective genes from both parents.
He was the first to link genes with enzyme production.
Garrod termed these defective genes “Inborn Errors of Metabolism”, and began the
field of Human or Medical Genetics.
Beadle and Tatum saw Garrod’s work while working on the nutritional needs
of a mold Neurospora.
After exposing mold spores to X-rays (to produce mutations) they isolated a
number of mutant strains which could not produce certain nutrients.
They were working on the inheritance or genetics of these mutant strains.
The metabolic pathway for Arginine production was known to be controlled by three enzymes.
They had several strains of Neurospora which could not produce Arginine.
They found that different strains had different mutations which all prevented
Arginine synthesis, but at different steps in the pathway.
They first proposed that each gene controlled a separate enzyme
– the “one gene, one enzyme” theory.
The One Gene – One Enzyme theory has been modified over time:
1. Genes do control the production of enzymes, but not all genes encode enzymes.
2. Not all enzymes consist of a single polypeptide. Some multimeric enzymes
are formed by the product of multiple genes, each producing a
separate polypeptide which combine to form the enzyme.
One Gene – One Polypeptide
3. Some genes are alternatively spliced to produce multiple proteins, or
versions of a protein. So some genes produce multiple proteins.
20,000 human genes produce some 2 million proteins.
The "Central Dogma" of Biology: traces the path of information flow within cells.
Transcription: Is DNA-Directed RNA Synthesis.
RNA Polymerase – the one molecule concerned with Transcription.
Promoter - region of gene where RNA Polymerase attaches to Transcribe gene.
Three types of RNA:
mRNA - carries the information (message) from DNA to the Ribosome.
rRNA - a component of Ribosomes.
tRNA - "escorts" amino acids to the Ribosome.
All 3 are Transcribed in the same way.
Ribonucleoside Triphosphates – precursors for Transcription.
Mechanism – removal of pyrophosphate and polymerization of Nucleotide to 3’ end
of growing RNA chain.
Transcription occurs on Template strand only.
Transcription occurs on one gene only.
RNA Polymerase attaches to DNA and denatures DNA.
In Eukaryotes, a variety of proteins are involved in this attachment.
They are termed Initiation and Transcription Factors.
Ribonucleoside Triphosphates line up opposite DNA Template strand and RNA
Polymerase connects them together in the 5’→3’ direction.
Within a chromosome, different strands of the double stranded DNA form the Template Strand.
Termination occurs in a variety of ways in different RNAs.
The final product (Primary Transcript), however, is not the finished product.
Post-Transcriptional Modification
RNAs are all Post-transcriptionally modified in a variety of ways.
1. The addition of a 5’ 7-methyl Guanosine “Cap” occurs in mRNA only.
2. Addition of a Poly-A “Tail” also occurs in mRNA only.
3. Splicing - The removal of introns, can occur to any type of RNA, from the action of
Spliceosomes a complex of RNA and Proteins.
The removal of introns results in an RNA Transcript much shorter than the
corresponding gene (DNA)
Alternative Splicing:
Not all introns are always included in the final mRNA.
If different introns are used to produce a mRNA, different proteins may be produced
in different cells or tissues from the same gene.
Some 20,000 human genes are used to produce some 2 million proteins.
4. The Primary Transcript may be cut into more than one RNA. (all RNAs
exhibit this modification, but it always occurs in rRNA).
5. The removal of nucleotides from the 5’ and/or 3’ end (occurs in all RNAs).
6. The chemical modification of bases occurs in tRNA only.
7. The addition of CCA to 3’ end (if already does not end with CCA) (occurs in tRNA only).
8. RNA Editing is a process by which one or more nucleotides in an RNA can
be changed, added, or removed.
In Substitution Editing, one nucleotide is substituted for another.
In Insertion/Deletion Editing, nucleotides are added or removed.
With Substitution Editing, enzymes modify or change one base into another.
Cytidine deaminases converts a C in the RNA to uracil (U).
Adenosine deaminase converts an A to inosine (I), which the ribosome Translates as a G.
Insertion/Deletion Editing uses guide RNAs produced by separate genes, to bind and
identify other RNA molecules in which a nucleotide will be added or removed.
RNA Editing has been found in three major types of RNA.all
Errors in RNA Editing can cause mental diseases such as schizophrenia.
Translation
All three RNAs are used for Translation:
mRNA carries the information
tRNA brings the amino acids
rRNA is a component of Ribosomes
mRNA: The Genetic Code
Triplet Code: 3 bases make up a Codon.
Codon - a sequence of 3 bases in mRNA which code for an Amino Acid.
There are 3 codons (Terminators) which stop Translation rather than insert an Amino Acid.
The codon AUG is the Initiation Codon, it marks the point where Translation begins.
The Genetic Code is degenerate (redundant), most Amino Acids are represented
by more than one Codon.
Transfer RNA
2D Shape - 3-leaf clover
Acceptor Stem - Amino Acid is attached to the 3' end of tRNA.
Transfer RNA - 3D shape of tRNA is L-shaped.
The Anticodon Loop is across from the acceptor stem, it pairs with the codon on mRNA.
Aminoacyl-tRNA Synthtases “Load" proper Amino Acid onto tRNA using energy from ATP.
First, the Aminoacyl tRNA Synthetase binds ATP and the specific Amino Acid
recognized by that Activating Enzyme.
The energy from ATP is transferred to the Amino Acid as AMP.
Then the specific tRNA recognized by the Synthetase is bound and the Amino
Acid transferred from the AMP to the tRNA.
Finally the tRNA, charged with the appropriate Amino Acid is released, as is the
Enzyme (in an unaltered state).
Ribosomes
Structure- Large and small subunits each constructed of one or more rRNA and 20-30 Proteins.
Self-assembly - the parts automatically come together to form Ribosomes.
Ribosome:
Has 3 positions to hold tRNAs:
1. A site – holds new tRNA.
2. P site – holds tRNA with polypeptide chain.
3. E site – holds empty tRNA to exit the ribosome.
RNA-Directed Polypeptide Synthesis
1. small ribosomal subunit attaches to 5’ end of mRNA.
2. first tRNA attaches to small subunit P Site.
Uses GTP as an energy source.
3. Large ribosomal subunit attaches to form the complete Ribosome.
4. The first Aminoacyl-tRNA has attached at the P-Site.
The 2nd aminoacyl-tRNA attaches at the A-site of the Ribosome, matching the
2nd codon of the mRNA.
The mRNA-Ribosome-tRNA Complex positions the Amino Acids so they
are adjacent to one another.
5. Peptidyl Transferase catalyzes a peptide bond between the first two
Amino Acids by transferring the first amino acid from the first tRNA to
the second amino acid.
6. Translocation moves the Ribosome to the next codon, using GTP as an energy source,
with release of first tRNA from the exit (E) site. The first Amino Acid is
attached to the second, which is attached to the second tRNA.
7. The 3rd aminoacyl-tRNA anticodon attaches to the A-site of the Ribosome
according to the 3rd mRNA codon.
8. Peptidyl Transferase catalyzes the next peptide bond. The Ribosome Translocates
in the 3’ direction once more.
9. Repeat of steps 6-8.
10. Upon reaching a termination (stop) codon, a Releasing Protein attaches
instead of an Aminoacyl tRNA.
The Releasing Factor binds to the A-Site and causes the release of the last tRNA and
the Polypeptide.
The two Ribosomal Subunits are released from the mRNA and are recycled to find
the same or another mRNA to Translate.
Each mRNA is Translated by numerous Ribosomes at the same time.
Once the first clears the 5’ end, the next attaches.
A Polysome is one mRNA and all the multiple Ribosomes Translating it at the same time.
Once synthesized, proteins with transit sequences may be exported to various organelles.
The Role of the Endoplasmic Reticulum in Glycoprotein Synthesis.
Signal Sequence – causes the Ribosome to be transported to the ER.
When completed, the Glycoprotein is released in the lumen of the ER and Ribosomal
Subunits are released to the Cytoplasm.
Post-Translational Modifications:
Once synthesized, proteins may be modified in a variety of ways:
Proteolysis Glycosylation Phosphorylation
