Transcript transcription and translationCellBio

E. coli lysed to release chromosome
Fig. 14.4 a
General overview of bacteria
• One of the three major lineages of life
– Eukaryotes – organisms whose cells have encased nuclei
– Prokaryotes – lack a nuclear membrane
• Archea
– 1996 complete genome of Methanococcus jannaschii sequenced
– More than 50% of genes completely different than bacteria and eukaryotes
– Of those that are similar, genes for replication, transcription, and translation are
same as eukaryotes
– Genes for survival in unusual habitats similar to some bacteria
• Bacteria
– Similar genome structure, morphology, and mechanisms of gene transfer to
archea
– Evolutionary biologist believe earliest single celled organism,
probably prokaryote existed 3.5 billion years ago
Diversity of bacteria
• Outnumber all other organisms on Earth
• 10,000 species identified
– Smallest – 200 nanometers in diameter
– Largest – 500 micrometers in length (10 billion
times larger than the smallest bacteria)
– Habitats range from land, aquatic, to parasitic
• Remarkable metabolic diversity allows
them to live almost anywhere
Common features of bacteria
•
•
•
•
Lack defined nuclear membrane
Lack membrane bound organelles
Chromosomes fold to form a nucleoid body
Membrane encloses cells with mesosome which
serves as a source of new membranes during cell
division
• Most have a cell wall
• Mucus like coating called a capsule
• Many move by flagella
•
Power of bacterial genetics is the
potential
to
study
rare
events
Bacteria multiply rapidly
– Liquid media – E. coli grow to concentration of 109 cells per milliliter
within a day
– Agar media – single bacteria will multiply to 107 – 108 cells in less
than a day
• Most studies focus on E. coli
–
–
–
–
Inhabitant of intestines in warm blooded animals
Grows without oxygen
Strains in laboratory are not pathogenic
Prototrphic – makes all the enzymes it needs for amino acid and
nucleotide synthesis
– Grows on minimal media containing glucose as the only carbon source
– Divides about once every hour in minimal media and every 20 minutes
in enriched media
– Rapid multiplication make it possible to observe very rare genetic
events
How to identify mutations by a
genetic screen
• Genetic screens provide a way to observe
mutations that occur very rarely such as
spontaneous mutations (1 in 106 to 1 in 108 cells)
– Replica plating – simultaneous transfer of thousands of
colonies from one plate to another
– Treatments with mutagens – increase frequency of
mutations
– Enrichment procedures – increase the proportion of
mutant cells by killing wild-type cells
– Testing for visible mutants on a petri plate
Fig. 7.12a1
Fig. 7.12a2
Fig. 7.12c1
Fig. 7.12c2
Fig. 7.12b1
Fig. 7.12b2
Fig. 8.11a
TRANSCRIPTION
Transcription occurs when RNA polymerase catalyzes the 5' to
3' synthesis of an RNA molecule – a fully processive process.
RNA synthesis is driven by the potential energy stored in
nucleotide triphosphates and is based on matching
complementary base pairs to the sequence in a template DNA
strand, started at a promoter in response to signals of cell needs.
You should be able to diagram (1) how transcription initiation,
elongation, and termination occur in bacteria and eukaryotes and
(2) how mRNAs are processed in eukaryotes.
Transcription Initiation:
Eukaryotic Genes in Pieces:
In eukaryotes, complex
machines do the splicing
out of the introns;
in the mitochondria of
tetrahymena and in phage,
it is done by ribozymes
that are actually part of
the intron itself. There,
no proteins are involved.
In phage, the introns also
encode so-called “homing
endonucleases”.
Caps and polyA tails on eukaryotic mRNAs are
involved in ribosome binding and messenger
stability:
Some of the similarities and differences observed in
transcription in bacteria versus eukaryotes:
How does a triplet of nucleotides specify a particular amino acid?
All of the specificity of
the translational process
is determined by the AAtRNA synthase. If one
puts an amino acid on
and then chemically
alters it to a different AA
it will get inserted into
the protein as if it were
that initial amino acid.
There are several very
different families of AAtRNA synthases that
probably evolved
seperately.
How Many tRNAs Are There?
• There are 61 different codons but only 40
or so tRNAs in most cells.
•The wobble hypothesis proposes that the
third position of the codon can form a
nonstandard base pair with the base
opposite it in the tRNA anticodon.
•Thus, one tRNA is able to base-pair with
more than one type of codon.
Many bacteria encode a suppressor tRNA that reads UGA instead of the normal
codon for that particular amino acid and occasionally suppresses a nonsense (socalled amber) mutation. These were first found using T4 phage.
Is the Ribosome an Enzyme or a Ribozyme?
• Ribosomes contain both a number of proteins and
ribosomal RNA (rRNA) and can be separated into two
subunits, the large subunit and the small subunit.
•Three-dimensional models completed in the year 2000
revealed that the active site consists entirely of
ribosomal RNA.
•Protein synthesis is catalyzed by RNA. The ribosome is
a ribozyme—not an enzyme.
tRNAs are found on 3 sites
in ribosomes:
The A site is the acceptor site
for the new AA-tRNA that binds
to the mRNA codon.
The P site holds the growing
polypeptide chain. As a peptide
bond forms between the amino
acid on the AA-tRNA in the A
site and the growing
polypeptide held on the tRNA
in the P site, the new A-site
tRNA moves to the P site.
The E site is where now-free
tRNAs exit the ribosome.
Initiation
• Translation begins at the AUG start codon.
•In bacteria, this codon is preceded by a ribosome
binding site (also called the Shine-Dalgarno
sequence, SD) that is complementary to a short
section of the 16s rRNA, in the small ribosomal
subunit.
•In eukaryotes, this is the first AUG of a capped
mRNA.
•In bacteria, several proteins can be encoded
sequentially along the same mRNA, coordinating
their production in time and space.
•The SD site has some subset of the sequence 5'-AGGAGGU-3'
and is located about six nucleotides upstream of the start codon.
• Once the small ribosomal subunit is bound to the mRNA,
the aminoacyl initiator tRNA binds to the AUG sequence.
•The methionine on this initiator tRNA has formic acid
(COOH) attached to its amino group and is called Nformylmethionine.
•The large subunit binds and completes the initiation
complex with the initiator tRNA is located in the P site of
the ribosome.
•This is possible because the attached COOH blocks the
positive charge of the amino group as if it were already part
of a peptide chain.
•The formyl group, and sometimes the whole formyl
methionine, is cleaved off after the protein is made.
E. coli lysed to release chromosome
Fig. 14.4 a
General overview of bacteria
• One of the three major lineages of life
– Eukaryotes – organisms whose cells have encased nuclei
– Prokaryotes – lack a nuclear membrane
• Archea
– 1996 complete genome of Methanococcus jannaschii sequenced
– More than 50% of genes completely different than bacteria and eukaryotes
– Of those that are similar, genes for replication, transcription, and translation are
same as eukaryotes
– Genes for survival in unusual habitats similar to some bacteria
• Bacteria
– Similar genome structure, morphology, and mechanisms of gene transfer to
archea
– Evolutionary biologist believe earliest single celled organism,
probably prokaryote existed 3.5 billion years ago
Diversity of bacteria
• Outnumber all other organisms on Earth
• 10,000 species identified
– Smallest – 200 nanometers in diameter
– Largest – 500 micrometers in length (10 billion
times larger than the smallest bacteria)
– Habitats range from land, aquatic, to parasitic
• Remarkable metabolic diversity allows
them to live almost anywhere
Common features of bacteria
•
•
•
•
Lack defined nuclear membrane
Lack membrane bound organelles
Chromosomes fold to form a nucleoid body
Membrane encloses cells with mesosome which
serves as a source of new membranes during cell
division
• Most have a cell wall
• Mucus like coating called a capsule
• Many move by flagella
•
Power of bacterial genetics is the
potential
to
study
rare
events
Bacteria multiply rapidly
– Liquid media – E. coli grow to concentration of 109 cells per milliliter
within a day
– Agar media – single bacteria will multiply to 107 – 108 cells in less
than a day
• Most studies focus on E. coli
–
–
–
–
Inhabitant of intestines in warm blooded animals
Grows without oxygen
Strains in laboratory are not pathogenic
Prototrphic – makes all the enzymes it needs for amino acid and
nucleotide synthesis
– Grows on minimal media containing glucose as the only carbon source
– Divides about once every hour in minimal media and every 20 minutes
in enriched media
– Rapid multiplication make it possible to observe very rare genetic
events
How to identify mutations by a
genetic screen
• Genetic screens provide a way to observe
mutations that occur very rarely such as
spontaneous mutations (1 in 106 to 1 in 108 cells)
– Replica plating – simultaneous transfer of thousands of
colonies from one plate to another
– Treatments with mutagens – increase frequency of
mutations
– Enrichment procedures – increase the proportion of
mutant cells by killing wild-type cells
– Testing for visible mutants on a petri plate