Lecture 5 Bacterial Genome Replication, expression, and regulation 1 Flashcards
All DNA present in a cell or virus
Genome
Bacteria and Archaea generally have ___ set(s) of DNA
1 (haploid-1N)
Eukaryotes have ___ set(s) of DNA
2 (diploid-2N)
Specific set of genes an organism possesses
Genotype
Collection of observable characteristics
Phenotype
Griffith in 1928 observed the change of non-virulent organisms into virulent ones as a result of ______
transformation
MacLeod and McCarty in 1944 showed that the transforming particle in Griffith’s experiments was ___
DNA
Explain how Hersey and Chase experimented to show DNA as a genetic material in 1952
- They used bacteriophage T2 infection as a model
- They labeled DNA with 32P (radioactive marker)
- They labeled the protein coat with 35S(radioactive marker)
- Their experiment showed that only DNA entered the cell but both new DNA and protein coats were synthesized in new viruses thus indicating DNA had genetic information for both of these viral components
DNA and RNA are polymers of nucleotides linked together by ______
phosphodiester bonds
DNA is transcribed into
- tRNA, mRNA, rRNA, snRNA
what are the forms of RNA
tRNA, mRNA, rRNA, microRNA, siRNA, snRNA
___ and ___ grooves form when the 2 DNA strands twist around each other
major and minor
Most RNA molecules are ____ stranded but some are _____
single, double stranded
Proteins are polymers of ____ linked by ____ bonds
amino acids, peptide
amino acids have a ___ carbon, ____ group, ___ group, and ___ chain
central carbon, carboxyl group (c-terminus), Amino group (N-terminus), and side chain
what determines the properties of the amino acids
the side chain
DNA replication is semi-conservative meaning what
each daughter cell obtains one old and one new strand
DNA in most bacteria is ____
circular
The portion of the genome that contains an origin that is replicated as a unit
replicon
Because the bacterial chromosome is a single replicon
The forks meet on the other side and two separate chromosomes are released
DNA in bacteria has _____ replication from a single origin
Bidirectional
the replication of Bacteria, such as E. coli, consists at least _____ proteins
30
The two replication forks in bacterial DNA replication proceed bidirectionally until they meet a site called the
replication termination site (ter)
What serve as DNA polymerase substrates
deoxynucleoside triphosphates: dATP, dTTP, dCTP, dGTP
What are incorporated into the growing DNA chain
deoxynucleoside monophosphates: dNMPs: dAMP, dTMP, dCMP, dGMP
For DNA polymerase to catalyze the synthesis of DNA it needs what 3 things
- A template (which is read in the 3’ to 5’ direction
- a primer (to provide a free 3’-hydroxyl group to which nucleotides can be added
- dNTPs
function of DnaA in E. coli
initiation of replication; binds origin of replication (oriC)
function of DnaB in E. coli
Helicase (5’–>3’) ; breaks hydrogen bonds holding two strands of double helix together ; promotes DNA primase activity; involved in promise assembly
function of DNA gyrase in E. coli
Relieves supercoiling of DNA produced as DNA strands are separated by helicases; separates daughter molecules in final stages of replication
Function of SSB (single stranded binding) proteins
Bind single-stranded DNA after strands are separated by helicases
Function of DnaC in E. coli
Helicase loader, helps direct DnaB protein (helicase) to DNA template
function of DNA primase
Synthesis of RNA; component of primosome
Function of DNA polymerase III holoenzyme
complex of about 20 polypeptides; catalyzes most of the DNA synthesis that occurs during DNA replication; has 3’—>5’ exonuclease (proofreading) activity
Function of DNA polymerase I
Removes RNA primers; fills gaps in DNA formed by removal of RNA primer
Functions of Ribonuclease H in E. coli
Removes RNA primers
Functions of DNA ligase
Selas nicked DNA, joining DNA fragments together
Function of Tus in E. coli
Termination of replication
What is the function of Topoisomerase IV in E. coli
Separation of chromosomes upon completion of DNA replication
E. coli has ____ different DNA polymerases
5
DNA polymerase III holoenzyme is a multifunctional enzyme composed of ___ different proteins
10
Polymerase III has ____ core enzymes
2 (some evidence suggests 3)
What are the functions of the 2 core enzymes in DNA polymerase III
- catalyze DNA synthesis (alpha subunit)
- Proofreading for fidelity (epsilon subunit)
what is the function of the tau dimer in DNA polymerase III holoenzyme
clamp loader, each subunit is attached to a core enzyme
_____ unwinds DNA strands
Helicases (DnaB)
Keeps strands apart for replication to occur
single stranded binding proteins (SSB)
Breaks one strand of DNA to relieve tension from rapid unwinding of double helix and prevents super coiling
Topoisomerases
_____ is an important topoisomerase in E. coli that is not only important during DNA replication but also for introducing negative supercoiling in the bacterial chromosome that helps compact it
DNA gyrase
synthesizes short complementary strands of RNA (about 10 nucleotides) to serve as primers needed by DNA polymerase
Primase (this is an RNA polymerase), which means that it can initiate RNA synthesis without an existing 3’-OH
The complex of primase and its accessory proteins is called the
primosome
Bacterial initiator protein ____ is responsible for triggering DNA replication
DnaA
DnaA proteins bind regions in ____ throughout the cell cycle, to initiate replication
OriC
After most of the lagging strand has been synthesized by the formation of Okazaki fragments. DNA polymerase ____ removes the RNA primers.
I
unlike DNA polymerase III DNA polymerase I has the ability to snip off nucleotides one at a time starting at the
5’ end while moving toward the 3’ end of the RNA primer
5’ to 3’ exonuclease activity
In the lagging strand at the end of each okazaki fragment the B clamp loader
is discarded and a new one is added
DNA ligase forms a phosphodiester bond between _________ of the growing strand and the ______ of an okazaki fragment
3’-hydroxyl, 5’-phosphate
DNA polymerase III has removes mismatched bases by _____ activity
3’-to-5’ exonuclease activity
Replication of E. coli stops when the replisome reaches _______ on DNA
termination site (ter)
____ form when the two circular daughter chromosomes do not separate after replication (interlocked)
Catenanes
____ is when Two chromosomes joined together to form a single chromosome that is twice as long
dimerized chromosome
Dimerized chromosomes are resolved by
XerCD recombinase (these catalyze an intramolecular cross-over that separates the two chromosomes
Catenanes arise from the activity of ______ during chromosome replication. Accordingly, they are resolved by _____
topoisomerases, topoisomerases
dimerized chromosomes arise from ______ that can occur during chromosome replication between daughter chromosomes
recombination events
how can bacteria solve the linear chromosome problem
- disguise their ends
- An enzyme telomere resolvase (ResT) forms hairpin ends for each daughter molecule
a basic unit of genetic information
Gene
codons are found in ____ and code for a single amino acid
mRNA
_____ strand of DNA directs RNA synthesis
template strand (read in the 3’-to-5’ direction)
____ DNA strand is the same nucleotide sequence as mRNA
coding strand (complementary DNA strand)
____ is the binding site in a gene for RNA polymerase
promoter
The promotor is neither transcribed nor translated; it functions
strictly to orient RNA polymerase so it is a specific distance from the first DNA nucleotide that will serve as a template for RNA synthesis
The transcription start site represents the
first nucleotide in the mRNA synthesized from the gene (note that this site does not necessarily code for amino acids)
What is the important sequence in the leader region ( point that is transcribed into mRNA but is not translated into amino acids) in bacteria that initiates translation
Shine-Dalgarno sequence
The coding region typically begins with the template DNA sequence _____
3’-TAC-5’ (this is transcribed into 5’-AUG-3’ which codes for the first amino acid)
3’-AUG-5’ on mRNA codes for
N-Formylmethione (start codon)
The coding region ends with
a stop codon immediately followed by a trailer (which is transcribed but not translated and prepares the RNA polymerase to detach), and then a terminator which makes the RNA polymerase detach
DNA sequences that code for tRNA and rRNA are considered ____
genes
Can genes coding for tRNA may code for more than a single tRNA molecule or type of tRNA
yes
genes coding for rRNA are transcribed as
single, large precursor
spacers between the coding region of both, tRNA and rRNA, are removed after _____, some by the use of special ribonuclease called _____
transcription, ribozymes
_____ carries amino acids during protein synthesis
tRNA
RNA has a complementary sequence to the ____ DNA
template
______ mRNA is often found in bacteria and archaea, while humans only have _____ mRNA
polycistronic, monocistronic
_______ has no catalytic activity but helps RNA polymerase holoenzyme recognize the promotor and initiate trancription
sigma factor
Transcription involves three separate processes
- initiation
- elongation
- termination
RNA polymerase is composed of ____ chains and catalyzes RNA synthesis
5
RNA polymerase RNA is
core enzyme + sigma factor (note that only the haloenzme can begin transcription)
bacterial promotors have two characteristic features, which are
a sequence of six bases about 35 base pairs before (upstream) the transcription starting point and a TATAAT sequence called the Pribnow box (about 10 base pairs before the starting site)
The Pribnow box is usually about _______ upstream of the transcriptional start site
10 base pairs upstream
sigma factor70 recognizes what consensus sequences
promotors having -10 and -35 sequences
what are consensus sequences
the promotor (ex. -10 and -35) that are recognized by a specific sigma factor (different sigmas mean different initiation sites)
After binding, RAN polymerase unwinds the DNA and a transcription bubble is produced, inside the bubble is
RNA:DNA hybrid
Some Transcription terminators require the aid of the ____ factor for termination
rho
There are how many codons
64
how many sense codons are there (codons that specify amino acids)
61
how many stop (nonsense) codons are there
3
RNA polymerase specifically binds to what part of the promotor sequence
Pribnow box
The Sine-Dalgarno sequence is in what region of what strand and marks what process
Leader of mRNA, signals the start of translation
What DNA sequence marks the start of transcription
3’-TAC-5’ this produces the codon AUG which codes for N-formylmethionine, a modified amino acid used to initiate protein synthesis in bacteria
DNA sequences that code for tRNA and rRNA are considered ____
genes
genes coding for tRNA may code for __________
more than a single tRNA molecule or type of tRNA
genes coding for rRNA are transcribed as
single, large precursor
Site where RNA polymerase binds to initiate transcription, is not transcribed, and has specific sequence before transcription starting point and a Pribnow box which contains consensus sequence
Promoter
Transcription termination
- occurs when core RNA polymerase dissociates from template DNA
- DNA sequence mark the end of gene in the trailer and the terminator
- soem terminators require the aid of the rho factor for termination
Code degeneracy
up to six different codons can code for a single amino acid
The ____ position of a codon is less important than the ___ or ___
3rd, is less important than the 1st or 2nd
some microbes incorporate two rare amino acids into polypeptides
- selenocysteine
- pyrrolysine
Is it true that some protist can use a single stop codon and have the other two code for amino acids
yes
what is the direction of translation
N terminus to C terminus
what is the site of translation
Ribosome
complex of mRNA with several ribosomes
polyribosome
coupled transcription/translation in _____
bacteria/archaea
attachment of amino acids to tRNA is catalyzed by _____
aminoacyl tRAN syntheses (at least 20)
The bacterial ribosome
- 70S ribosome= 30S and 50S subunits
- Translational domain on both subunits is responsible for translation
_____ rRNA ribosomal binding site (RBS) binds to shine dalgarno site on mRNA for protein synthesis
16S
what rRNA binds protein needed for initiation of translation and amino acyl-t-RNA
16S rRNA ribosomal binding site (RBS)
Ribozyme catalyzes peptide bond formation at what rRNA
23S rRNA
Bacterial initiator tRNA
N-formylmethionine-tRNA
Archaea and eukaryote initiator tRNA
Methionine-tRNA
in bateria initiation of translation begins when
- initiator codon binds 16S rRNA in 30S subunit
- Shine Dalgarno sequence of mRNA is aligned with 16S rRNA
_____ initiation factors (IF) in bacteria are required for the formation of the initiation complex of translation
3 (note that the reaction is catalyzed by GTP)
Sequential addition of amino acids to growing polypeptide
Elongation cycle
The elongation of the polypeptide chain consists of three phases what are they
- aminoacyl-tRNA binding
- transpeptidation reaction
- translocation
Binds initiator tRNA or tRNA attached to growing polypeptide (peptidyl-tRNA)
Peptidyl (donor; P ) site
Binds incoming aminoacyl-tRNA
aminoacyl (acceptor; A) site
briefly binds empty tRNA before it leaves ribosome
exit (E) site
Transpeptidation reaction is catalyzed by
peptidyl transferase of 23S rRNA
In transpeptidation reaction the amino group of the ____ site amino acid reacts with the carboxyl group of the C-terminal amino acid on the ____ site tRNA. Thus the peptide chain is
A, P, the peptide chain is transferred from P site to A site
What are the 3 simultaneous evens in the final phase of elongation cycle- Translocation
- peptidyl-tRNA moves from A site to P site
- ribosome moves down one codon
- empty tRNA leaves P site
(note that this requires GTP hydrolysis)
what are the three stop (nonsense) codons
UAA, UAG, UGA
_____ aid in the recognition of stop codons
release factors (RFs)
How many Release factors are in prokaryotes
3
how many release factors are in eukaryotes
1
Protein function depends on 3-D shape and this occurs as a post translational event including
- requires folding
- association with other proteins
- delivered to proper sub cellular or extracellular site.
Proteins that aid the folding of nascent polypeptides
molecular chaperones
Functions of molecular chaperones
- proteins that aid the folding of nascent polypeptides
- protect cells form thermal damage
- aid in transport of proteins across membranes
movement of proteins from cytoplasm to plasma membrane or periplasmic space
Translocation (include transport proteins, ETC proteins, proteins involved in chemotaxis and cell wall synthesis, enzymes)
Movement of proteins from the cytoplasm to external environment
Secretion
major pathway for all bacteria for transporting proteins across the plasma membrane
Sec-dependent pathway
Gram-negative bacteria common translocation and secretion systems
- Proteins can be transported across the outer membrane by several different mechanisms, some of which bypass the Sec system, by moving proteins directly from the cytoplasm to the outside of the cell.
- may use sec-dependent pathway b
- also must cross the outer membrane using Types I, II, III,IV, V systems
all common translocation and secretion pathways require
energy
The sec-dependent pathway is also called the
General dependent pathway
The sec-dependent pathway is ____ conserved in all domains
Highly
posttranlational Proteins secreted by sec-dependent pathway are synthesized as preproteins having amino-terminal _____, which function is
Signal peptide, which is recognized and bound by chaperone proteins (SecB) this helps delay protein folding
______, ______, and ______ form a channel in the membrane for the sec-dependent pathway
- secY
- secE
- secG
_____ translocates preprotein through the plasma membrane
secA
in the sec pathway When the preprotein emerges through the plasma membrane a signal peptidase
removes the signal peptide and thus allows folding to take place
Type I secretion systems
- related to ABC transport systems
- Gram-positive/Gram-negative bacteria, and archaea
- Secretion of toxins, proteases, other proteins
Type I secretion systems are involved in the secretion of
toxins, proteases, and other proteins
What is the Tat system
- protein translocation system in bacteria and some archaea
- moves across plasma membrane
- Tat pathway translocated folded proteins with “twin” arginine residues in their signal sequence
- Works with Type II secretion system (in gram negative bacteria it uses Type II secretion system to transport proteins across the outer membrane)
how is the Tat system distinguished from the sec pathway
by the nature of the protein transported. The sec pathway translocates unfolded proteins; the Tat pathway translocates folded proteins. furthermore the TAT pathway only moves proteins that feature two, or “twin” arginine residues in their signal sequence (note tat stands for twin arginine translocase)
Type IV secretion system secretes ______ and ____ and is found in
- secretes proteins and secretes DNA from donor to recipient bacterium during conjugation
- found in both Gram-positive and Gram-negative
how many protein secretion systems are there in gram-negative bacteria. which ones are also found in gram-positive
6, Type I and IV are also found in gram-postive (note that the ones unique to gram-negative II,III, and V secrete virulence factors)
what is the function of Type II secretion pathway
transports proteins across the outer membrane that were first translocated across plasma membrane by sec-dependent pathway
Does Type I secretion pathway bypass the Sec pathway
yes it spans the periplasmic and outer-membrane
Type ___, ____, and ___ systems are able to transport proteins without help form the sec system (thus are sec independent)
Type I, III, and VI
Type III secretion pathway forms ______ and transports
injectisomes (needles) that deliver virulence factors and proteins
Why are type V secretion pathway called auto transporters
because after using the sec pathway to get across the periplasmic membrane they are able to form a pore and transport themselves through the outer membrane
what type of secretion pathway is similar to bacteriophage
Type VI
What are the two approaches to regulation of protein
- Regulation of gene expression (transcription initiation, elongation, and translation)
- Alter activity of enzymes and proteins (post translational)
what are constitutive genes
are housekeeping genes that are expressed continuously by the cell
What are inducible genes
genes that code for inducible enzymes needed only in certain environments
Beta-galatosidase reaction catalyzes
lactose hydrolysis into galactose and glucose
____ enzymes are present only when their substrate is available
inducer
enzymes that function in biosynthetic pathways are products of
repressible genes
many enzymes that function in catabolic pathways are ____ enzymes
inducible
Inhibiting transcription is ____ control
negative
promoting transcription is _____ control
positive
in bacteria repressor proteins attache to a region called the _____, which usually overlaps or is downstream of the promotor
operator
Activator proteins attach to ________, and these are often upstream of the promoter
Activator-binding sites
what is the function of corepressors
they activate aporepressors
enzymes of a catabolic pathway are only needed (increased mRNA synthesis) when the
preferred substrate is available
The lactose operon is a ______ transcriptional control of inducible genes
negative
The lac operon contains ____ structural genes controlled by the ____, which binds the operator
3, lac repressor (lacl) (enzymes are not normally produced unless lactose is present)
tetramers of lac repressor form and bind to ____ operator sites
3 (O1, O2, O3)
Lac repressor bends DNA and prevents _____ from accessing promoter
RNA polymerase
Presence of ____ binds to lac repressor ( thus meaning it is no longer bound to operator)
allolactose
what is the secondary regulatory protein of the lac operon
catabolite activator protein (CAP)
CAP regulates the lac operon in response to the presence or absence
of glucose.
The highest levels of transcription of the lac operon occur when lactose is ____ and Glucose is
available and glucose is not
The lowest levels of transcription of the lac operon occur when lactose is ____ and glucose is
not available, and glucose is
what do the genes of the lac operon produce
- beta-galactosidase
- Beta-galactoside permease (note this is the protein responsible for lactose uptake)
- Beta-galatoside transacetylase
what is the main function of catabolite activator protein (CAP)
is an activator of the lac operon that is responsible for maintaining glucose levels
The tryptophan (trp) operon consists of ____ structural genes which code for enzymes needed to synthesize tryptophan
5
Negative transcriptional control of repressible genes of the tryptophan operon is controlled by
tryptophan repressor
is tryptophan repressor active when tryptophan levels are low
no
What is the corepressor for the tryptophan repressor
Trypotphan (increased levels lead to activation of trp repressor)
The arbinose operon has transcriptional control by a protein that acts
both positive and negatively
the area operon encodes enzymes needed for catabolism of
arbinose to xyluose 5-phosphate (an intermediate of the pentose phosphate pathway)
The ara operon is regulated by ____, which can interact with three different regulatory sequences: ___, ____ , and ____
AraC, araO2, araO1, and araI
The activity of AraC on the Arabinose (ara) operon depends on ___ . explain
Activity depends on environmental conditions. When arabinose is present it inactivates AraC and can actually open the DNA fro transcription. When arabinose is absent then AraC blocks the promotor and RNA synthase
what is attenuation
Termination of transcription within the leader region (leader peptide)
The ability to attenuate transcription is based on what two things
- the nature of the leader and the fact that transcription and translation are coupled in bacteria
attenuation was first demonstrated with
trp operon
attenuation occurs through _____
stem-loop structures in the mRNA depending on trp level
regulation by ______ is a specialized form of transcription attenuation that involves mRNA folding but not ribosome behavior
riboswitches (sensory RNAs)
explain how riboswitches regulate transcription
if the leader of mRNA (the riboswitch) is folded one way than transcription continues if it is folded another way then it is terminated. (note that the mRNA folding pattern is determined by binding an effector molecule directly to the mRNA- a capability previously thought confined to proteins
Controlling transcription with riboswitches is an important method used by _______ to regulate amino acid-related genes
Gram-positive bacteria
