Chapter 15 – Genes and how they work

15.1/ The Nature of Genes

- George Beadle and Edward Tatum, 1941
- they studied Neurospora crassa (fungus)
- They used X-rays to damage the DNA in cells of Neurospora (spores)
- they looked for fungal cells lacking specific enzymes due to genetic mutations
- the enzymes were required for the biochemical pathway producing the amino acid arginine
- they identified mutants deficient in each enzyme of the pathway
Beadle and tatum proposed that each enzyme of the arginine pathway was encoded by a
separate gene.
One gene – one enzyme hypothesis
One gene – one polypeptide hypothesis

Conversion of genotype to phenotype:

The central dogma of molecular biology states that information flows in one direction
DNA  RNA  protein
Transcription is the flow of information from DNA to RNA
Translation is the flow of information from RNA to protein

Gene expression overview:

Template strand: strand of the DNA double helix used to make RNA
Coding strand: strand of DNA that is complementary to the template strand
RNA polymerase: the enzyme that synthesizes RNA from the DNA template

Transcription proceeds through:

- Initiation: RNA polymerase identifies where to begin transcription
 promoter, enzyme -RNA polymerase-, transcription factors , start site
- Elongation: RNA nucleotides are added to the 3’ end of the new RNA
 5’ to 3’ synthesis of messenger RNA (mRNA)
- Termination: RNA polymerase stops transcription when it encounters terminators in the
DNA sequence.
 terminators

RNA has multiple roles in gene expression

All RNAs are synthesized from a DNA template by transcription. Gene expression requires
the participation of multiple kinds of RNA, each with different roles in the overall process.
Here is a brief summary of these roles, which are described in detail in the remainder of the
chapter.
- Messenger RNA: even before the details of gene expression were unraveled, geneticists
recognized that there must be an intermediate form of the information in DNA that can
be transported out of the eukaryotic nucleus to the cytoplasm for ribosomal processing.
This hypothesis was called the “messenger hypothesis”, and we retain this language in
the name messenger RNA (mRNA).
 - Ribosomal RNA. The class of RNA found in ribosomes is called ribosomal RNA (rRNA).
There are multiple forms of rRNA, and rRNA is found in both ribosomal subunits. This
rRNA is critical to the function of the ribosome.
- Transfer RNA. The intermediary adapter molecule between mRNA and amino acids is
transfer RNA (tRNA). Transfer RNA molecules have amino acids covalently attached to
one end and an anticodon that can base-pair with an mRNA codon at the other. The
tRNAs act to interpret information in mRNA and to help position the amino acids on the
ribosome.
- Small nuclear RNA. snRNAs are part of the machinery that is involved in nuclear
processing of eukaryotic “pre-mRNA”.
- SRP RNA. In eukaryotes, where some proteins are synthesized by ribosomes on the
rough endoplasmic reticulum (RER), this process is mediated by the signal recognition
particle, or SRP. The SRP contains both RNA and proteins.
- Small RNAs. This class of RNA includes both microRNA (miRNA) and small interfering
RNA (siRNA). These are involved in the control of gene expression.

15.3/ Prokaryotic Transcription

Prokaryotic cells contain a single type of RNA polymerase found in 2 forms.
- Core polymerase is capable of RNA elongation but not initiation
- Holoenzyme is composed of the core enzyme and the sigma factor which is required for
transcription initiation
Initiation of transcription:
A transcriptional unit extends from the promoter to the terminator.
The promoter determines the site of initiation and the direction of transcription and is
composed of:
- A 6 base-pair DNA sequence TTGACA for the binding of RNA polymerase located at – 35
base-pairs and recognized by the sigma subunit factor
- A 6 base-pair sequence TATAAT at – 10 base-pairs also recognized by the sigma subunit
where the helix is opened.
The start site (+1) = the first base to be transcribed
Initiation of synthesis with ATP or GTP
 5’ to 3’ synthesis

Elongation of transcription:
- Clearance of enzyme = dissociation from promoter by conformational changes
- During elongation, the transcription bubble moves down the DNA template at a rate of
50 nucleotides/sec
the transcription bubble consists of:
- RNA polymerase
- DNA template
- Growing RNA transcript
Hybrid DNA-RNA (9bp) = stabilize the 3’ end of growing RNA
Termination occurs at specific sites
The end of bacterial transcription unit is marked by terminator sequences that signal “stop” to
the polymerase. Reaching these sequences causes the formation of phosphodiester bonds to
cease, the RNA-DNA hybrid within the transcription bubble to dissociate, the RNA polymerase
to release the DNA, and the DNA within the transcription bubble to rewind.
- Transcription stops when the transcription bubble encounters terminator sequences
- This often includes a series of G-C repeats followed by a series of A-T pairs (4 or more
Us)
- Hairpin formation = the RNA transcript of this stop region can form double-stranded
structure in the GC regions called hairpin, which is followed by 4 or more uracil.
- Formation of the hairpin causes the RNA polymerase to pause, placing it directly over
the run of four uracils. The pairing of U with the DNA’s A is the weakest of the four-
hybrid base-pairs, and it is not strong enough to hold the hybrid strands when the
polymerase pauses. Instead, the RNA strand dissociates from the DNA within the
transcription bubble, and transcription stops. A variety of protein factors also act at
these terminators to aid in terminating transcription.
 in prokaryotes, transcription and translation are often coupled = occurring at the same time
- In prokaryotes, mRNA produced may contain multiple genes encoding related functions
in a single transcriptional unit = operon.
- An operon is a single transcription unit that encodes multiple enzymes necessary for a
biochemical pathway. When genes are clustered by function, they can be regulated
together.

15.4/ Eukaryotic transcription

Eukaryotes have three RNA polymerases
RNA polymerase I  transcribes rRNA
RNA polymerase II  transcribes mRNA and some smaller nuclear RNAs
RNA polymerase III  transcribes tRNA and some other small RNAs
 together, these three enzymes accomplish all transcription in eukaryotic cells

Each RNA polymerase recognizes its own promoter

- rRNA genes have species-specific promoters
- mRNA genes have most complex and diverse promoters composed of several elements
including a “core element” = TATA box (but not all)
- tRNA promoters are internal to the genes = located in introns
15.7/ The Process of Translation
Initiation of translation:
Initiation of transcription is more complex
It requires a series of transcription factors = proteins that act to bind RNA polymerase to the
promoter and initiate transcription hence forming an initiation complex

Eukaryotic pre-mRNA’s modification

In eukaryotes, the primary transcript or pre-mRNA must be processed/modified to form a
mature mRNA by:
1- addition of a 5’ cap
 addition of a methylated GTP by a 5’-5’ bond = methyl G-cap
 protection of mRNA from degradation and role in translation initiation
2- addition of a 3’ poly-A tail (1-200amino acids)
 after a cleavage site (AAUAAA)
 poly-A polymerase
 role in stability of mRNA and protection against degradation
3- removal of non-coding sequences or introns by splicing

Eukaryotic pre-mRNA splicing

Eukaryotic genes (unlike in prokaryotes) contain:
- introns – non-coding sequences
- exons – coding sequences that will be translated
The spliceosome is the organelle responsible for removing introns and splicing exons together
Small ribonucleoprotein particles (snRNPs) within the spliceosome recognize the intron-exon
boundaries
 the processes of transcription, pre-mRNA modifications and splicing all form one
integrated/concerted process to produce the mature mRNA

15.2/ The genetic code

Deciphering the genetic code: how 4 nucleotides (A, T, C, G) could encode more than 20 amino
acids?
Francis Crick and Sydney Brenner determined that the DNA is read in sets of 3 nucleotides for
each amino acid
Chemically induced mutations to a viral DNA molecule:
A. deletion of a single/double/triple nucleotide
B. additions of a single/double/triple nucleotide
Codon = set of 3 nucleotides that specify a particular amino acid
Reading frame = the series of nucleotide read in sets of 3 (codon)
Reading is continuous without punctuation or spaces between codons
Only one reading frame is correct for encoding the correct sequence of amino acids
Frameshift mutations = alter reading frame of the genetic message
Marshall Nirenberg identified the codons that specify each amino acid -genetic code-
 in vitro using free systems
 synthetic RNA molecules
- poly-U RNA molecule codes for the polypeptide poly-phenylalanine
- RNA polymers with two or three different nucleotides

Gobin Khorana
Synthetic RNA molecules of defined sequence
64 possible three nucleotides sequences and their corresponding amino acids

Stop codons: 3 codons (UAA, UGA, UAG) in the genetic code use to terminate translation
Start codon: the codon (AUG) used to signify the start of translation coding for methionine

The code is degenerate meaning that some amino acids are specified by more than one codon
but the opposite is not true.
- Degeneracy is not uniform
- Degeneracy is the third base pair
The code is universal meaning it is the same in all organisms
- Common evolutionary origin of species
- Exception in mitochondria and chloroplasts (‘stop’ signals)

tRNA:
tRNA adapter molecules carry amino acids to the ribosome incorporation into a polypeptide
tRNA are bifunctional molecules with a highly conserved structure in all living organisms:
- An amino acid attached to the acceptor arm or acceptor stem in the 3’ end of tRNA –
5’CCA3’-
- The anticodon loop contains 3 nucleotides complementary to mRNA codon
The enzyme called aminoacyl-tRNA synthase charges the amino acids into tRNA
- It recognizes many tRNA but only one amino acid
- The reaction is called “charging reaction” and joins the COOH end of an amino acid to
the free OH in the acceptor stem (3’) for form a charged amino acid (activated
intermediate)

Ribosomes
The ribosomes consist of two subunits made of rRNA (functional components) that are held
together by proteins (supporting components)
- Small subunit = decoding function (along with tRNA)
- Large subunit = contains the peptidyl transferase that catalyzes the polypeptide
synthesis or formation of peptide bonds
The ribosome has multiple tRNA binding sites arranged 5’ to 3’:
- A site – (aminoacyl) binds the tRNA carrying the next amino acid
- P site – (peptidyl) binds the tRNA attached to the growing peptide chain
- E site – (exit) binds the tRNA that carried the last amino acid
 tRNA moves through these sites successively during translation starting with A, P then E.
Translation:
Translation proceeds in three steps:
1- Initiation: a number of initiation factors mediate this interaction of the ribosome, mRNA,
and tRNA to form the initiation complex. These factors are involved in initiation only and
are not part of the ribosome.
2- Elongation: when the entire ribosome is assembled around the initiation tRNA and
mRNA, the second charged tRNA can be brought to the ribosome and bind to the empty
A site. This requires elongation factor. A peptide bond can then form between the
amino acid of the initiator tRNA and the newly arrived charged tRNA in the A site. The
geometry of this bond relative to the two charged tRNAS is critical to understanding the
process. Remember that an amino acid is attached to a tRNA by its carboxyl terminus.
The peptide bond is formed between the amino end of the incoming amino acid (in the
A site) and the carboxyl end of the growing chain (in the P site).
Step 1: matching tRNA anticodon with mRNA codon
Step 2: peptide bond formation
Step 3: translocation of the ribosome
3- Termination: ribosome encounters a stop codon and releases polypeptide
 stop codon, release factors

Initiation of translation
In prokaryotes, initiation of translation requires the formation of the initiation complex
including:
- An initiator tRNA charged with N-formyl methionine (P site)
- The small ribosomal subunit
- mRNA strand
- initiation factors
the ribosome binding sequence is a conserved sequence in the 5’ end of the mRNA that is
complementary to part of rRNA in the small subunit.
 the initiation in eukaryotes is similar to prokaryotes but with few exceptions:
- initiator amino acid is charged with methionine
- initiation complex is more complicated (9 or more factors)
- the small ribosomal subunit binds to the 5’ cap of mRNA

Elongation of translation
Elongation of translation involves the addition of amino acids in cycles:
- matching tRNA anticodon with mRNA codon on site A: a charged tRNA binds to the A
site
- peptide bond formation by peptidyl transferase
- ribosome translocation: the ribosome moves down the mRNA in a 5’ to 3’ direction
The peptide bond is formed between the amino acid end (NH3) of the incoming amino acid in
site A and the carboxyl group COOH end of the growing chain in site P.
Two GTP molecules are hydrolyzed: one after the binding of the new charged tRNA to site A
and the second one during the ribosome translocation
The tRNAs move through the ribosome in opposite direction compared to the ribosome, that is
from site A to P then finally E before being ejected and recharged again.
 Wobble pairing: as mentioned, there are fewer tRNAs than codons. This situation is easily
rationalized because the pairing between the 3’ base of the codon and the 5’ base of the
anticodon is less stringent than normal. In some tRNAs, the presence of modified bases with
less accurate pairing in the 5’ position of the anticodon enhances this flexibility. This effect is
referred to as wobble pairing because these tRNAs can “wobble” a bit on the mRNA, so that a
single tRNA can “read” more than one codon in the mRNA.

Termination of translation
Elongation continues until the ribosome encounters a stop codon
Stop codons are recognized by release factors which release the polypeptide from the ribosome

Synthesis of proteins by ribosomes on the RER

In eukaryotes, translation may occur on ribosomes in the cytoplasm or on ribosomes of the
RER.
Signal sequences are found at the beginning of the polypeptide sequence of proteins destined
to the RER.
The signal sequences are recognized by a cytoplasmic complex called the signal recognition
particle (SRP)
The signal sequence and SRP are in turn recognized by receptor proteins in the RER membrane.
The signal sequence/SRP holds the ribosome on the RER; a process called “docking”. As the
polypeptide is synthesized it passes through a channel into the interior of the endoplasmic
reticulum.

Mutation: altered genes

Point mutation = alter a single base in the DNA sequence
- base substitution mutation  substitutes one base for another = transitions or
transversions
- missense mutations  a base substitution mutation may not change the encoded
amino acid. If it does not, we say the mutation is synonymous or silent. If it changes an
amino acid, we say the mutation is nonsynonymous, also referred to as missense
mutations.
- Silent mutations
- Nonsense mutations  a mutation that changes a codon from one that encodes an
amino acid to a stop codon called a nonsense mutation because the codon can no
longer make “sense” to the translation apparatus. The stop codon will cause translation
to terminate prematurely, producing a truncated protein.
- Frameshift mutations  cause by insertion or deletion of a single base/ shifts reading
frame / premature termination
Triplet repeat expansion mutation
- Involves a sequence of 3 DNA nucleotides that are repeated many times
- The abnormal allele causing the disease contains theses repeats whereas the normal
allele does not
- Triplet repeats are associated with some human genetic diseases for example,
Huntington disease, fragile X-syndrome…

Chromosomal mutations
Change the structure of chromosomes
- Deletion – part of chromosome is lost
- Duplication – part of chromosome is copied
- Inversion – part of chromosome in reverse order
- Translocation – part of chromosome is moved to a new location

 too much genetic mutations can be harmful to the individual/ species. However, genetic
variation (caused by mutations) is necessary for evolutionary change of the species.