From gene to protein

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From gene to protein Again, insights into the
connection between DNA and protein were made
before the molecular connection was
established Garrod- inborn errors of
metabolism Beadle and Tatum- one gene, one
enzyme hypothesis (in modern terminology- one
gene one polypeptide)
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RNA is the link between genes (DNA) and
proteins Transcription the synthesis of mRNA,
using the DNA sense strand as a
template mRNA provides the sequences for protein
synthesis translation
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Significant differences between processes
in eukaryotes and prokaryotes Nucleus Ribosome
structure RNA processing
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The genetic code consists of nucleotide
triplets Encoded in mRNA Specified by
DNA Discovered in the 1960s 64 possible codons
61 specify amino acids Nonoverlapping Reading
frame must be correct
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Genetic code is nearly universal Some
microorganisms use different codons E. coli and
yeast have been modified so that they can add
modified amino acids to a polypeptide Bacteria
can make them yeast can take them up from
environment (in other words, codons recognize
the novel amino acids)
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RNA polymerase forms the mRNA molecule Promoter
guides polymerase binding terminator stops
it Prokaryotes have one RNA polymerase eukaryot
es have three
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RNA polymerase I- makes precursors for ribosomal
RNAs (except for smallest subunit) RNA
polymerase II- mRNA and snRNAs (involved in RNA
processing) RNA polymerase III- variety of RNAs
smallest rRNA subunit, tRNA precursors Each
uses a different promoter (DNA sequences that
direct polymerase to begin tran- scribing
there) Promoters are upstream from coding
sequence
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Polymerases also use transcription factors Bind
in a specified order, either to promoter or each
other RNA polymerase (II) must be
phosphorylated before it can start synthesizing
RNA
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A gene can be transcribed by many polymerases
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Elongation factors required to open up
DNA molecule so polymerase can move Each
polymerase has its own termination signals too
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Termination signal AAUAAA Pre-mRNA is released
from enzyme Poly(A) tail is added Helps
stabilize RNA 5 cap has similar function also
helps bind ribosomes
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What is RNA processing? All modifications
necessary to generate final RNA product Removal
of portions of primary transcript Addition or
modification of specific nucleotides (such as
methylation) Location of RNA to cytoplasm
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What about rRNA and tRNA? rRNA is most abundant
(70-80 of all cellular RNA) and the most
stable Four types in eukaryotes, three in
prokaryotes transcription units contain the
genes for the rRNAs separated by
spacers Spacers are transcribed too, but
degraded later RNA polymerase I is utilized in
nucleoli of eukaryotes
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After rRNA is processed it assembles
with proteins to form ribosomal subunits Many
different tRNA molecules are made all are small
(70-90 nucleotides) In processing many of these
are chemically modified Some of these, and
most mRNAs, contain introns
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What is an intron? A sequence within the primary
transcript that is not found in the mature
RNA (noncoding DNA sequence) Exons describe the
sequences that do contain coding
information Some genes have a few introns,
others many
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Examples Human ?-globin has two introns Human
dystropin (largest human gene currently known)-
2 million bp and 78 introns! Are introns
actually transcribed? Yes, introns were by
looking at mixtures of DNA and mRNA, and DNA and
pre-RNA
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How are the introns removed?
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Splice sites are contained in the ends of
introns
Cuts out intron Joins exons together
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Why are there introns? With a few exceptions,
most are simply cut out of preRNA transcripts
and degraded Allows for alternative splicing of
RNAs (joining of different exons
together) 35,000 human genes produce mRNAs
coding for over 100,000 polypeptides
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Evolutionary significance of introns May have
helped foster new combinations of exons to yield
new proteins Exon shuffling Duplication (and
later mutation) of exons
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RNA editing Nucleotides may be inserted, deleted
or modified and can drastically change the
message Seen in mitochondria of protozoans,
plants Probably rare
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rRNA and tRNA are stable, but mRNA is
not (therefore most transcription is of
mRNA) Affects how much protein is made by a
cell many copies of mRNA can be made from a
single DNA sequence many copies of mRNA?many
polypeptides many copies of genes for rRNA and
tRNA cant amplify those!