Download READ: Protein Synthesis File

TRANSCRIPTION AND TRANSLATON
Gene Expression
Transcription and translation are the two fundamental components of gene
expression. These two processes are part of the transformation of genetic
information into functional molecules. Transcription makes an RNA-based copy
of a DNA sequence. Translation makes an amino-acid polymer based on an RNA
sequence. Transcription and translation are usually coupled: transcription occurs
first and translation follows. The information flow, from DNA to RNA via
transcription, and then from RNA to protein via translation, is one-way. Cells do
not make RNA sequences from amino-acid templates or DNA sequences from
RNA templates.
TRANSCRIPTION

Transcription requires DNA: Prokaryotic DNA is located in the cytoplasm,
so prokaryotic transcription occurs in the cytoplasm. In eukaryotes, the DNA
is located in the nucleus, so eukaryotic transcription occurs in the nucleus.
Mitochondria and chloroplasts have their own DNA, and transcription can
also occur in each of these structures.

Base Pairs: The nucleotides in RNA form base pairs with the nucleotides in
DNA. RNA has four different nucleotides: adenine (A), cytosine (C), guanine
(G), and uracil (U). DNA has the same nucleotides except for U, which is
replaced by thymine (T) in DNA. Each nucleotide pairs exclusively with one
of the others. G always pairs with C. T, which only occurs in DNA, pairs with
A. In RNA, A pairs with U.

RNA Polymerase: Transcription requires an enzyme called RNA
polymerase. RNA polymerase links nucleotides together to make an RNA
that is complementary to a pre-existing piece of DNA. The DNA must be
uncoiled for the RNA polymerase to access its bases, and the hydrogen
bonds between the base pairs of the DNA double helix must be broken.
The template strand consists of the DNA strand that forms complementary
base pairs with the nascent RNA. The coding strand is the DNA strand that
is complementary to the template strand. The RNA polymerase always
moves along the template strand in a 3’ to 5’ direction. The nucleotide
sequence in the newly synthesized RNA is the same as the nucleotide
sequence in the coding strand, except the Ts are replaced by Us in the
RNA.

Promoters: DNA sequences called promoters specify the location on the
DNA strand where the RNA polymerase should start transcription. The
promoters are upstream. This means that they are further toward the 3’ end
of the template DNA than from the first nucleotide that is actually
transcribed. In bacteria, the RNA polymerase binds to the promoters with
the help of an accessory protein called sigma factor. In Archaea and
eukaryotes, the RNA polymerase does not actually bind to the promoters. In
these organisms, proteins called transcription factors are what bind to the
promoters. After the transcription factors bind to the promoters, the RNA
polymerase binds to the DNA and transcription begins. Once transcription
initiates, the sigma factor (transcription factors) dissociate from the DNA,
and the RNA polymerase moves along the template strand in a 3’ to 5’
direction, synthesizing a new RNA molecule. Transcription ends when the
RNA polymerase reaches sequences in the DNA that cause the enzyme to
dissociate from the DNA, either with or without the help of additional
transcription-termination factors. The end result of transcription is a singlestranded RNA copy of a segment of the coding strand of the DNA.

Types of RNA: Most of the RNAs synthesized via transcription are
messenger RNAs (mRNAs). mRNAs serve as templates for protein
synthesis. Not all RNAs are mRNAs. Ribosomal RNAs (rRNAs) function as
components of ribosomes, or the RNA-protein complexes that synthesize
proteins. Transfer RNAs (tRNAs) function as adapters to link specific amino
acids with specific codons during translation. Other small RNAs function by
binding through complementary base pairing to mRNAs, preventing their
translation, or to sites within the DNA, enhancing or suppressing
transcription. Small regulatory RNAs, rRNAs, and tRNAs do not encode
proteins. These specific RNAs are non-coding, functional RNAs
TRANSLATION
Translation makes a polypeptide chain based in an mRNA. Translation occurs
on ribosomes, which are located within the cytoplasm and on cytoplasmic
membranes. Ribosomes match amino acids to nucleotide triplets,
called codonsin the mRNA sequence. Each codon specifies a certain amino
acid. The mapping of amino acids to their codons is called the genetic code. The
genetic code is fairly consistent across all of the domains of life, with many slight
variations. Mitochondria and some bacteria, for example, use slightly modified
genetic codes.

Condons and Anticodons: Ribosomes synthesize polypeptides through
the successive formation of peptide bonds between amino acids.
Ribosomes match amino acids to codons by bringing tRNAs together with
mRNAs. tRNAs carry a specific amino acid at one end, and an anticodon at
the other end. An anticodon is a nucleotide triplet that is complementary to a
codon. For example, the anticodon for the GGG codon is CCC. The GGG
codon specifies the amino acid glycine, so the corresponding tRNA has a
glycine at one end and the CCC anticodon at the other end. When the
ribosome reaches the GGG codon in the mRNA, it matches the codon to the
CCC anticodon in a glycine tRNA, then adds the glycine at the other end of
the tRNA to the growing polypeptide chain. The tRNA moves out of the
ribosome after its amino acid joins the growing polypeptide, and the mRNA
moves a distance of three nucleotides, or one codon, through the ribosome.
Thus, during translation, the mRNA moves through the ribosome in threenucleotide increments. Each time the mRNA moves ahead, a new tRNA
enters the ribosome, adds its amino acid to the growing polypeptide, and
then leaves the ribosome.

Mapping the Genetic Code: As there are four different nucleotides, and
three nucleotides make up one codon, there are 64 possible codons. Three
of the 64 codons are stop codons. The stop codons do not code for an
amino acid, but instead signal the point at which translation should stop.
Each of the other 61 codons specifies an amino acid. There are only 20
standard amino acids, so most amino acids have more than one codon.

Thus, the genetic code is redundant and also degenerate. Degeneracy
refers to the fact that certain positions within certain codons can be changed
without changing the amino acid. For example, the codons AAU and AAC
both code for the amino acid asparagine. The third position in both of the
asparagine codons is 2-fold degenerate, which means that two different
nucleotides have the same meaning. The codons CUU, CUA, CUC, and
CUG all code for the amino acid leucine, so the third position in these
codons is 4-fold degenerate, as four different nucleotides have the same
meaning.
Polypeptides: The end result of translation is a polypeptide chain in which
the sequence of amino acids matches the corresponding sequence of
codons in an mRNA. The new polypeptide undergoes a complex folding
process, often with the help of molecular chaperones and other posttranslational modifications, to finally become an active protein. A single
mRNA can be used to synthesize multiple proteins with multiple ribosomes
simultaneously translating the mRNA. Multiple RNA polymerases can also
simultaneously transcribe mRNAs from a single gene by proceeding, one
after another, along the DNA strand.
REGULATORY NETWORK
Transcription and translation are tightly regulated and controlled. No cell
expresses all of its genes all of the time. For transcription, the region of DNA
containing a gene must be accessible to transcription factors and RNA
polymerases. Furthermore, the DNA cannot be bound by histones and must be
mostly linear (not coiled). Enzymes called helicases unwind the DNA double
helix. Regulatory proteins and small RNAs interact with non-coding sequences
within the DNA to direct the conformational changes as well as the binding of
helicases and transcription factors. Other non-coding DNA sequences called
enhancers are not necessary for transcription, but their presence upstream or
downstream from a gene can affect the rate of transcription. Protein expression
is also regulated post-transcriptionally. Post-translational modifications and
protein folding can be enhanced or suppressed, resulting in higher or lower levels
of active proteins within the cell.
Through intricate regulatory networks, cells turn genes on and off, depending on
changing needs and conditions. The total number of genes is not very different
between humans and single-celled organisms. Much of the difference between
complex organisms and simpler ones is due to differences in the transcriptional
regulatory networks rather than differences in the number of genes that they
posses. The different proteins that are synthesized at different times determine
the characteristics of cells and the organisms that they make up.
GENETIC DISORDERS
Genetic disorders arise from mutations in DNA sequences. For a genetic
disorder to pass from one generation to the next in sexually reproducing
organisms, the mutation must be carried by the gametes (sperm or eggs).
Mutations in the somatic cells can cause problems for the individual in which they
occur, but the offspring will not be affected as they inherit only the DNA carried
by the gametes. Mutagens are physical or chemical agents that cause
mutations. Mutations within protein-coding regions of DNA can cause problems if
they are not repaired as they may result in dysfunctional proteins, especially if
the mutation causes a frame-shift or a premature stop codon. Mutations outside
of coding regions can also cause problems if they occur within promoters,
enhancers, or other regulatory sites. A mutation that destroys a promoter can
stop a protein from being transcribed, even though the coding region of the gene
remains unchanged. Mutations are related to cancer, as they change gene
expression and alter the properties of proteins.

Point mutations are single-nucleotide changes. A point mutation within a
coding region changes a codon, which may or may not result in a different
amino acid in the corresponding polypeptide. A point mutation in a coding
region that does not change the amino acid that is encoded is called a
synonymous substitution, whereas one that changes the amino acid is
called a non-synonymous substitution.


Insertions and deletions: Insertions and deletions are the addition or
removal of pieces of DNA within a chromosome. The size of insertions and
deletions can range from a single nucleotide to entire chromosomes.These
mutations can change the reading frame of a coding sequence. As the
genetic code is read three nucleotides at a time, adding or removing a
number of bases (other than by a factor of three) changes every codon after
the mutation. For example, if the original nucleotide sequence is
AAAGGGCCCAAA, then the codons are AAA, GGG, CCC, and AAA. If a
single-base deletion changes the nucleotide sequence to AAGGGCCCAAA,
then the codons become AAG, GGC, and CCA.
Frame-Shift Mutation: A mutation that causes a change in the reading
frame of a gene is called a frame-shift mutation. Frame-shift mutations are
devastating as the resulting protein looks nothing like the un-mutated
version, and is likely not to function at all. Mutations resulting in a premature
stop codon are also highly disruptive. Any of the three stop codons will
cause translation to terminate. For example, an mRNA with the sequence
CACUGGUCU has the codons CAC, UGG, and UCU, which will make a
polypeptide with histidine, tryptophan, and serine. If a mutation changes the
sequence to CACUGAUCU, then the second codon becomes UGA, which
stops translation after the histidine. A mutation resulting in a premature stop
codon is usually referred to as a knockout (unless it is near the end of the
gene) as it effectively removes the corresponding protein from production.
Diploid organisms have two copies of each chromosome, so a knockout of
one copy may not completely remove
the protein from production. However,
having only a single working copy of a
gene may still have serious
consequences. The cell may not be able
to produce the protein at a high enough
rate from only one chromosome, or the
working copy of the gene may have
some other mutation that causes it to function poorly.