微生物遺傳與生物技術
(Microbial Genetics and
Biotechnology)
金門大學
食品科學系
何國傑 教授
Bacterial gene expression
and its regulation
I. Terminology
1. Gene: DNA region that carry the information for synthesis of
RNA or RNA and protein.
2. Codon: each 3 nucleotides on DNA or RNA.
3. Genetic code: The assignment of each of the possible codons
to amino acids.
4. The first step in gene expression is to transcribe (or copy) an
RNA from one strand of DNA.
5. Types of RNA: There are many different types of RNA in cells.
The major three type are mRNA (messenger RNA), rRNA
(ribosomal RNA) and tRNA (transfer RNA).
(1) mRNA – The RNA carries the gene’s messenger (or
information) for protein.
(2) rRNA – RNA component of a ribosome.
(3) tRNA – The RNA carries an amino acid to a codon on mRNA.
II. Structure of RNA
1. RNA is similar to DNA in that it is composed of a chain of
nucleotides
2. Nucleotides of RNA contain the sugar ribose instead of
deoxyribose. The second carbon of ribose is attached to a
hydroxyl group rather than hydrogen in deoxyribose.
3. RNA has uracil instead of thymine found in DNA.
4. RNA is usually single stranded. However, a secondary
structure can form by pairing between the bases in some
regions of molecule may cause it to fold up on itself to form
double-stranded region.
5. The rule for double-stranded RNA is slightly different from
pairing rule for DNA. In RNA G can pair with U as well as C.
GU does not share hydrogen bond, it does not contribute to
the stability of the double-stranded RNA.
II. Structure of RNA
II. Structure of RNA
6. RNA can form tertiary structure when the unpaired region in a
hairpin pairs with another region of the same RNA molecule to
form a knot, a pseudoknot.
7. RNA processing and modification
(1) In RNA processing, the covalent bonds can be broken and
the smaller pieces of RNA can be religated into new
recombination. One of the most extreme cases of RNA
processing, called RNA editing, the nucleotides can be
excised or added to mRNA after it has been from DNA.
(2) RNA modification involved altering the bases or sugar of
RNA. For example, the methylation of bases and sugars.
II. Structure of RNA
II. Structure of RNA
Ψ: pseudouracil
III. Transcription
• The synthesis of RNA on DNA template and is work of RNA
polymerase
1. In eukaryotic cells, there are three kinds of RNA polymerases:
RNA polymerase I for rRNA synthesis; RNA polymerase II for
mRNA synthesis; RNA polymerase III for tRNA synthesis.
In prokaryotic cells such as bacteria, there is only one kind of
RNA polymerase for all three type of RNA synthesis, exception
that the RNA synthesis of the primer for Okazaki fragment.
2. E. coli RNA polymerase consists of six subunits: two identical
α, one β, one β’, one small ω and σ factor.
3. α and β subunits are essential parts of RNA polymerase; ω
helps the assembly of RNA polymerase and σ factor is
required for initiation and cycles of the enzyme after initiation
of transcription. RNA polymerase without σ factor is called
core enzyme, and with it is called holoenzyme.
III. Transcription
• One of σdomain, σ2 contacts β’ subunit and is in position to bind to the -10
region of the promoter.
• Another two domains, σ3 and σ4 contact the β subunit further upstream in the
active-center channel in such a way that domain σ4 in position to contact the -35
region of promoter.
*β and β’ form pincers of crab claw
III. Transcription
4. Much like DNA polymerase, RNA polymerase makes a
complementary copy of a DNA template, building a chain of
RNA by attaching the 5’ phosphate of a ribonucleotide to the 3’
hydroxyl of the one preceding it.
(1) RNA polymerase does not need a preexisting primer to
initiate the synthesis of RNA chain.
(2) Firstly, RNA polymerase binds to a specific region of DNA,
called promoter, and separate DNA to expose the bases
(Fig. 2.6).
(3) RNA polymerase recognizes different types of promoters on
the basis of which type of σ factor is attached.
(4) Even promoters of the same type are not identical to each
other, but they do share certain sequence, called consensus
sequences by which they can distinguish.
(5) A promoter sequence has two important regions: a short
AT-rich region about 10 bases upstream of transcription start
site, called – 10 sequence or TATA box, and a region about
35 base upstream of start site, called -35 sequence.
III. Transcription
III. Transcription
III. Transcription
III. Transcription
(6) RNA polymerase recognizes a particular T or C in the
promoter region as a transcription start site and assigned
as +1.
5. Initiation and elongation of transcription
(1) Core enzyme of RNA polymerase may be randomly bound to
DNA.
(2) A σ factor binds to the core enzyme of RNA polymerase
and then the holoenzyme recognizes and binds to a
promoter.
(3) When RNA polymerase binds to promoter, a closed complex
is formed because the DNA is still double-stranded.
(4) The DNA is melt at -10 region and forms a open complex.
(5) In the initiation process, a single nucleoside triphosphate
(usually A or G) enters and pairs with nucleotide (usually T
or C) at +1 in the template strand.
III. Transcription
III. Transcription
(6) Then a second nucleoside triphosphate enters and a
phosphodiester bond forms between its α phosphate and
the 3’ hydroxyl of ribose in the first nucleotide, releasing
two phosphates in the form of pyrophosphate and form an
initial transcription complex.
(7) At this stage, RNA polymerase is not yet free to continue the
transcription. A short RNA of about 10 nucleotides is
released. This abortive transcription occurs to various
degrees on many promoters until the RNA leaves the
promoter. (for proofreading)
i. Because when the RNA chain grows to a length of about
10 nucleotides, it encounters the σ3.2 loop in active-site
channel blocking the exit, called the exit channel. This
causes the release of transcript, a phenomenon of
abortive transcription.
III. Transcription
(8) Eventually, a growing transcript ( at least 12 nucleotides in
length) pushes the aside and enters the exit channel,
causing the factor to be released from the core RNA
polymerase.
(9) Once the RNA polymerase has initiated transcription at a
promoter, it continues along the DNA , polymerizing
ribonucleotides (a process called elongation), until it
encounters a transcription termination site on DNA. (Even
after transcription is under way, the polymerase often
pauses and sometimes even backs up before continuing
(for proofreading).
III. Transcription
III. Transcription
III. Transcription
Antibiotic rifampin can bind to the wall of active site channel and
prevent further elongation of RNA.
6.Termination of transcription
(1) Bacterial DNA has two basic types of transcription termination
sites: Factor (rho, ρ) dependent and factor independent.
i. Factor-dependent termination:
(i) Factor-dependent terminators have very little sequence
in common with each other and so are not readily
apparent. Fig. 2.19 illustrates a current model for
factor-dependent termination.
(ii) Theρfactor attaches to the mRNA at a rut (rho utilization)
site if the rut region is not being translated, and form a
hexameric ring around it.
(iii) The ρ factor moves along the mRNA with the cleavage
of ATP until it catches up with paused RNA polymerase
at a termination site (ρ-sensitive pause site). (ρ factor has
ATPase activity)
(iv) The helicase activity of ρ factor dissociates the RNA-DNA
hybrid in the transcription bubble, causing the
RNA polymerase and the RNA to be released.
A current model for factor-dependent
termination
6.Termination of transcription
ii. Factor-independent termination:
(i) Factor-independent terminators typically consist
an inverted repeat (GC-rich) followed by a short
string of A’s, usually 6 residues.
(ii) Fig. 2.19 illustrates a current model for factorindependent termination.
(iii) The U-rich RNA causes RNA polymerase to pause,
allowing a hairpin loop to form, dissociating RNA
polymerase and RNA.
A current model for factorindependent termination
III. Transcription
7. rRNA and tRNA synthesis
(1) Transcription of the genes for all the RNAs of the cell is
basically the same. rRNA and tRNA are synthesis as a
precursor, the individual rRNA and tRNA are cut from it. At
some point during the processing, the RNAs are modified to
make the mature rRNAs and tRNAs.
(2) rRNA
i. The structural component of ribosome, where the proteins
are synthesized.
ii. Bacterial ribosome contains three types of rRNA: 16S, 23S
and 5S. The 16S and 23S rRNA are made in a precursor
and then processed. (They are 18S, 28S and 5.8S in
eukaryotic cells).
iii. The rRNAs are among the most highly evolutionarily
conserved of all the cellular constituents. For this reason,
they are always used as the candidate for molecular
phylogeny analysis to clarify species.
III. Transcription
iv. In addition to their structural role in ribosome, the rRNAs also
play a direct role in translation: ex., The 23S rRNA is the
peptidetransferase (a kind of ribozyme). The 16S rRNA is
directly involved in both initiation and termination of
translation.
v. in many bacteria, the coding sequences for rRNAs are
repeated in 7 to 10 different places around the genome.
vi. The rRNAs sometimes are modified, for example methylated.
This modification sometimes confers resistance to some
antibiotics.
(3) tRNA (Fig. 2.21)
i. The tRNAs are probably the most highly processed and
modified RNAs in cells: Mature tRNA was cut from a much
longer molecule and some bases were modified by specific
enzymes, creating altered bases such as psedouracil and
thiouracil. An enzyme called CCA transferase added the
sequence CCA to the 3’ end of mature tRNAs.
The structure of rRNA precursor
The structures of tRNA
IV. Translation
@ Translate the sequence of nucleotides in mRNA into the
sequence of amino acids in protein, occurring on ribosome.
1. Ribosome consists of one copy each of the 16S, 23S, and 5S
rRNAs as well as over 50 different proteins.
2. The complete ribosome, called 70S ribosome, consists of two
subunits, the 30S subunit and 50S subunit. The 30S subunit
contains 16S rRNA and 21 different proteins. The 50S subunit
contains 23S rRNA and 31 different proteins.
3. Reading frame
(1) Each three nucleotide-sequence, or codon, in the mRNA
encodes a specific amino acid, and the assignment of the
codons is known as the genetic code.
(2) Translation begins at an initiator codon and ends at a
terminator codon, establishing a reading frame of
translation.
The structure of ribosome
IV. Translation
(3) Before translation can begin, a specific amino acid is attached
to nucleotide A of 3’ CCA sequence of each tRNA by its
cognate aminoacyl-tRNA synthetase.
(4) During translation, the ribosome moves three nucleotides at a
time along the mRNA in the 5’- to 3’- direction, allowing aatRNAs to pair with larger mRNA through codon-anticodon
pairing (Fig. 2.25, 2.26).
i. Actually, only two bases is sufficient to direct the codonanticodon interaction. In other word, codon-anticodon
paring is a wobble or degenerated. This pattern of
redundancy is due to the pairing between first base in
anticodon on the tRNA and last (third) base in the codon is
less stringent. As a consequence of wobble, the same
codon can have more than one tRNAs. These tRNAs are
called cognate tRNA, which carry the same amino acid
(Table 2.1).
Aminoacylation of a tRNA by its cognate
aminoacyl-tRNA synthetase
Complementary pairing between a tRNA
anticodon and an mRNA codon
Wobble pairing between a tRNA
anticodon and an mRNA codon
IV. Translation
4. Detail of translation
(1) Translation initiation
i. Initiation codon(s) – The initiation codon is usually
AUG, but in bacteria sometimes are GUG, UUG
or AUA. No matter which sequence, it specifies the amino
acid methionine (Met).
ii. mRNA has sequence called translational initiation region
(TIR), which contains an initiation codon and usually a
short sequence (~4 bases), called ribosomal binding site
(or Shine-Dalgarno sequence), upstream from the
initiation codon. In fact, the 5’ end of the mRNA may be
some distance from TIR. This region is called the 5’
untranslated region (5’UTR) or leader sequence.
IV. Translation
iii. Ribosomal binding site, also called Shine-Dalgarno (SD)
sequence is complementary to short sequence located at
3’ of 16S rRNA
iv. There are two sites in ribosome: A and P sites for aa-tRNA to
enter the ribosome and an E site for tRNA to exist from
ribosome.
v. Fig. 2.32 diagram the initiation of translation.
(i) Initiation factor IF3 binds the 30S subunit to keep it
dissociated from 50S subunit during initiation.
(ii) IF1 binds to A site to block this site.
(iii) The P site of 30S subunit binds the mRNA TIR site.
(iv) The formyl-Met (fMet) binds to initiation tRNA, tRNAfMet.
(v) The fMet-tRNAfMet enters the P site on 30S subunit in
the presence of IF2-GTP.
IV. Translation
(vi) IF1 and IF3 are released, the cleavage of GTP on IF2
correctly positions the fMet-tRNAfMet on the P site, and
the 50S subunit binds.
(vii) The 70S ribosome is ready to accept another aminoacyltRNA at A site.
(viii) Normally, polypeptides do not have a formyl group
attached to their N terminus, which is removed by
peptide deformylase after synthesized. In fact, they
usually do not even have methionine as their N terminal
amino acid, which is removed by methionine
aminopeptidase.
The pairing between SD sequence on mRNA
and a short sequence close to the 3’ end of
16S rRNA
Initiation of translation
Genetic code
Comparision between met and fMet
IV. Translation
(2) Translation elongation (Fig. 2.27)
i. When 70S ribosome forms, another aminoacyl-tRNA can
enter the A site.
(i) Translation elongation factor Tu (EF-Tu) binds another
aminoacyl-tRNA and helps position it in A site by using
the energy of GTP cleavage.
(ii) The Tu-GDP is phosphorylated to Tu-GTP by Ts-GTP,
another elongation factor.
ii. The peptidyltransferase then joins this coming amino acid
to the fMet at P site.
iii. The growing peptide is then move to P site by translation
elongation factor G (EF-G), making room at A site for
another aminoacyl-tRNA and using the energy of GTP
cleavage.
The peptidyltransferase reaction
Removal of formyl group and Nformyl-Met