Transcript gene expression… from DNA to protein

gene expression…
from DNA to protein
biology 1
• Genes control metabolism
• Gene expression is a two stage process
– transcription
– translation
• Genes consists of triplets of nucleotides
- the genetic code
• Protein synthesis in prokaryotes and
eukaryotes
– Eukaryotic modification of RNA
• Mutations
Genes control metabolism
• One gene-One polypeptide rule
• Polypeptides that are constructed as a
result of transcription/translation
process become either
– structural proteins
– enzymes
• Those proteins that have quaternary
structure may have polypeptides
originating from different genes
The transcription/translation process
• Transcription: DNA codes for the
construction of mRNA
• Translation: mRNA is read by rRNA at a
ribosome; tRNA brings amino acids to
ribosome as defined by code on mRNA
• Ribosome assembles polypeptide
Recap on RNA - a ribose nucleic acid that
uses Uracil (U) in place of Thymine (T)
The genetic code
• The linear sequence of nucleotides in DNA ultimately
determines the linear sequence of amino acids in a
polypeptide
• There are approximately 20 types of amino acid to choose
from
• In DNA, the four nucleotides are ATCG
• Therefore, the sequence of four possible nucleotides must
code for 20 amino acids
– If DNA used a individual nucleotide to refer to an individual amino
acid, this system would only code for 41 amino acids
– Using two nucleotides would account for 42 = 16 amino acids
– Using three nucleotides would account for 43 = 64 amino acids
• Since there are only 20 amino acids, yet 64 possible
codes, some redundancy occurs
• Each block of three nucleotides, ultimately
corresponding to a particular amino acid, is
called a codon
• In the first stage of the gene expression
process, transcription, the information in the
codons of a gene are transferred to mRNA
• This process is via an RNA polymerase that
uses one of the DNA strands of the double helix
(the template strand)
• For each amino acid, there are generally
several codons possible. Also, some codons
have a non-amino acid equivalent, but instead
send specific messages to RNA polymerase
(start/stop)
Transcription
• Three phases
– Polymerase binding and initiation
– Elongation
– Termination
• In eukaryotes, RNA polymerase II bind to
specific regions on DNA called promoters
• Promoters are typically 100 nucleotides long,
including
– The initiation site, where transcription begins
– Nucleotides sequences that help initiate
transcription
• Initiation in eukaryotes requires
transcription factors, DNA-binding
proteins that bind to specific nucleotide
sequences in the promoter region
– A common place for a transcription factor
to bind is the TATA box
– RNA polymerase recognizes the promoter
site once DNA and transcription factor
have bound at the TATA box
• RNA polymerase temporarily separates
the double helix for transcription
• In elongation, RNA polymerase II
(eukaryotes)
– Untwists the DNA molecule
– Adds incoming RNA free-floating nucleotides to
the 3’ end of the RNA strand (grows 5’ to 3’)
• mRNA grows at 30-60 nucleotides/sec. The
mRNA chain starts to peel away as the
double helix reforms
– Followed in series, several molecules of RNA
polymerase can simultaneously transcribe the
same gene
• Transcription proceeds until the polymerase
reaches a termination code
Translation
• During translation, proteins are synthesized
according to a genetic message of sequential codons
along mRNA
• tRNA (transfer RNA) interprets between the base
sequence in mRNA and the amino acid sequence in
a polypeptide chain. To do this…
– Transfer amino acids from cytoplasm to ribosome
– Recognize the correct codons on mRNA
• Molecules of tRNA are specific to one particular
amino acid
– One end of tRNA attaches to a specific amino acid (3’ end)
– The other end attaches to an mRNA codon by base pairing
with its anti-codon
• An anti-codon is a nucleotide triplet in tRNA
• tRNA decodes the genetic message codon by
codon
• There are 45 types of tRNA, which is
sufficient for the 64 codes, since there is a
relaxation of base-pairing on the third
nucleotide (wobble)
– e.g., U in 3rd position of anticodon can bind with
A or G on the equivalent codon
– In some cases, third position on a tRNA
anticodon is occupied by Inosine (a sixth
nucleotide) that can bind with U, C or A
• Joining of tRNA to specific amino acid at the 3’ end
is by Aminoacyl-tRNA synthetase
• Each amino acid has a particular synthetase
enzyme
– ATP activates the amino acid by losing 2 phosphate
groups, and joining to the amino acid as AMP
– tRNA bonds to the amino acid, which loses AMP
• Ribosomes coordinate the pairing of tRNA
anticodons to mRNA codons
– Consist of 2 subunits (small and large) that remain
separated when not involved in protein synthesis
– Ribosomes are composed of 60% rRNA and 40%
protein
• In addition to an mRNA binding site, two
further sites on a ribosome are the Pand A-sites
– P-site holds the tRNA carrying the growing
polypeptide chain
– A-site holds the tRNA that has the next
amino acid in the polypeptide sequence
• Building of a polypeptide chain consists
of three steps
– Initiation
– Elongation
– Termination
Translation Initiation
• In eukaryotes, the small ribosomal unit binds
to an initiator tRNA (methionine; anticodon
UAC)
• The small ribosomal unit binds to the 5’ end
of mRNA, and in doing so brings the tRNA
anticodon in close proximity with mRNA
methionine codon
• This binding requires initiation factors
• Finally, the large subunit binds to the complex
– The initiator tRNA fits to the p-site of the ribosome
– The vacant a-site is ready for the next aminoacyltRNA complex
Translation elongation
• Codon recognition—mRNA codon in the asite of the ribosome forms hydrogen bonds
with anti-codon of an entering tRNA carrying
the next amino acid in the chain
• Peptide bond formation—The enzyme
peptidyl transferase (part of the large
ribosomal unit) catalyzes the peptide bond
between the incoming amino acid and the
growing polypeptide chain
• Translocation—the tRNA in the p-site
releases from the ribosome, and the tRNA in
the a-site moves into the vacated site
Translation termination
• A termination codon signals the end of
translation; by binding to a protein
release factor, this causes:
– Peptidyl transferase hydrolyzes the bond
between the completed polypeptide and
the tRNA in the p-site
– This frees the polypeptide and tRNA so
that they can release from the ribosome
– The two ribosomal units disassociate
– mRNA may continue to be translated by
polyribosomes
Differences between prokaryotic
and eukaryotic gene expression
• Lack of nuclear membrane in prokaryotes means
that transcription can occur at one end of the
mRNA molecule, while translation can be occurring
at the other end
• In eukaryotes, RNA is modified following
transcription before translation
– 5’ cap added (modified guanine nucleotide
– Poly-A tail added (200 adenine nucleotides) to 3’ end
– These ends might protect mRNA sequence (attaching to
untranslated leader and trailer sequences respectively)
• Gene splicing
Gene splicing
• Eukaryotic mRNA has segments of non-code, called introns
(code sequences called exons)
– Introns and exons are initially coded into one long strand called hnRNA
(heterogenous RNA)
– In RNA splicing, introns are removed from hnRNA to make mRNA
• Process of splicing mRNA involves SnRNPs (“snurps”) - small
nuclear ribonucleoproteins, that are composed of SnRNA
(small nuclear RNA) and proteins
– Together with extra proteins, SnRNPs form complexes called
spliceosomes, which excise introns (SnRNPs attach to either end of
each intron)
– tRNA and rRNA also need to be spliced, but different agents do the
splicing - ribozymes, RNA molecules that act as enzymes (note: thus
not all enzymes are proteins)
• Why do introns exist?
– May regulate gene activity
– Splicing may regulate export of mRNA to
cytoplasm
– Introns cause exons to be further apart,
and therefore to be further away from each
other on the chromosome: this could mean
a higher probability of recombination during
cross-over
– Specific introns may code for specific
domains within a protein
When things go
wrong...
• Mutation = a permanent change in DNA
that can involve large chromosomal
regions or a single nucleotide pair
• Point mutation = a mutation limited to
one or two nucleotides in a single gene
– Base-pair substitution
• Missense mutation
• Nonsense mutation
– Insertion/deletion mutations
• Base-pair substitutions generally have
no effect if they occur on the third
nucleotide of a triplet
– If they do change the amino acid, one a.a.
substitution may not radically affect the
functionality of the final polypeptide
– In some cases, functionality is improved: in
most cases, functionality is impaired
– In nonsense mutations, the substitutions
causes a triplet to read STOP, abruptly
terminating polypeptide chain. Such
mutations are usually harmful
• Insertions or deletions add or remove
one or more nucleotides from a
sequence
– Since a reading frame for nucleotides is
based on a series of three, insertions and
deletions that add or remove a sequence
of nucleotides not divisible by 3 can
substantially alter the final polypeptide
– Such a mutation is referred to as a
frameshift - these mutations usually result
in non-functional proteins, unless they
occur towards the end of a sequence