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Summary of Past Questions
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- Microbiology
- Institution
- North-West University (NWU)
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MKBS Questions:Question 1:
Study Unite 1+2: Chapter 13
Events that take place at the replication fork during replication of Escherichia coli
1. Helicase unwinds parental DNA and one strand is coated with SSB proteins (lagging strand).
When a sufcient length of DNA is unwound, primase synthesizes an RNA primer. The clamp
loader then loads a clamp onto the template DNA to tether a core enzyme to the strand.
Lagging strand synthesis then begins.
2. Lagging strand synthesis is discontinuous and generates Okazaki fragments. Two core
enzymes function in lagging strand synthesis., although it is unknown if they do so
simultaneously as shown here
3. Notice that the lagging strand core enzyme on the left is approaching an already completed
Okazaki fragment. When the core enzyme reaches that fragment, the core enzyme will be
released from the DNA template.
4. The third core enzyme of DNA polymerase holoenzyme moves in the same direction as the
replication fork and synthesizes DNA continuously (leading strand synthesis)
OR According to lecture slide notes:
1. Initiation: DnaA proteins bind oriC (origin of replication; AT-rich) causing bending and
separation of strands
2. DnaB and other helicases separate strands, SSB attach; DNA gyrase removes supercoiling
3. Primase synthesizes RNA primer
4. DNA polymerase III holoenzyme: Leading and lagging strand is synthesized; Proofreading
5. DNA polymerase I or RNaseH removes RNA primers (5'-3' exonuclease activity); replace with
DNA
6. Okazaki fragments are joined by DNA ligase
7. Termination of replication, release of daughter chromosomes
,Give the steps happening at the replication fork in table format with the enzymes involved as well as
the function of each enzyme. Make sure you list it in the correct order
Role of bacterial promoters and sigma factors in transcription initiation.
- Each gene (or, in bacteria, each group of genes transcribed together) has its own promoter.
A promoter contains DNA sequences that let RNA polymerase or its helper proteins attach to
the DNA. Once the transcription bubble has formed, the polymerase can start transcribing.
A promoter is a region of DNA where transcription of a gene is initiated. Promoters are a
vital component of expression vectors because they control the binding of RNA polymerase
to DNA. RNA polymerase transcribes DNA to mRNA which is ultimately translated into a
functional protein. In bacteria, sigma factors are necessary for recognition of RNA
polymerase to the gene promoter site. The sigma factor allows the RNA polymerase to
properly bind to the promoter site and initiate transcription which will result in the
production of an mRNA molecule.
How many covalent hydrogen bonds occur between adenine and thymine on the one hand and
between guanine and cytosine on the other hand? Why are the hydrogen compounds important in
double-stranded DNA?
- The purine adenine (A) of one strand is always paired with the pyrimidine thymine (T) of the
opposite strand by two hydrogen bonds. The purine guanine (G) pairs with cytosine (C) by
three hydrogen bonds. Hydrogen bonds are weak, noncovalent interactions, but the large
number of hydrogen bonds between complementary base pairs in a DNA double
helix combine to provide great stability for the structure.
Discuss in detail the initiation of transcription in prokaryotes
- Transcription in prokaryotes (and in eukaryotes) requires the DNA double helix to partially
unwind in the region of mRNA synthesis. The region of unwinding is called a transcription
bubble. The DNA sequence onto which the proteins and enzymes involved in transcription
bind to initiate the process is called a promoter. In most cases, promoters exist upstream of
the genes they regulate. The specific sequence of a promoter is very important because it
, determines whether the corresponding gene is transcribed all of the time, some of the time,
or hardly at all. The structure and function of a prokaryotic promoter is relatively simple.
One important sequence in the prokaryotic promoter is located 10 bases before the
transcription start site (-10) and is commonly called the TATA box. To begin transcription,
the RNA polymerase holoenzyme assembles at the promoter. The dissociation of σ allows
the core enzyme to proceed along the DNA template, synthesizing mRNA by adding RNA
nucleotides according to the base pairing rules, similar to the way a new DNA molecule is
produced during DNA replication. Only one of the two DNA strands is transcribed. The
transcribed strand of DNA is called the template strand because it is the template for mRNA
production. The mRNA product is complementary to the template strand and is almost
identical to the other DNA strand, called the non-template strand, with the exception that
RNA contains a uracil (U) in place of the thymine (T) found in DNA. Like DNA polymerase,
RNA polymerase adds new nucleotides onto the 3′-OH group of the previous nucleotide. This
means that the growing mRNA strand is being synthesized in the 5′ to 3′ direction. Because
DNA is anti-parallel, this means that the RNA polymerase is moving in the 3′ to 5′ direction
down the template strand.
Function of the following in the transcription cycle in bacteria:
RNA polymerase core enzyme - completes RNA synthesis once it has been initiated.
Responsible for polymerization
Promoter sequence – The region on DNA at the start of a gene that RNA polymerase binds to
before beginning transcription
Sigma factor – A protein that helps bacterial RNA polymerase core enzyme recognize the
promoter at the start of a gene. Thus, it is a transcription factor
Rho protein – binds to the transcription terminator pause site, an exposed region of single
stranded RNA after the open reading frame at C-rich/G-poor sequences that lack obvious
secondary structure
Different steps in polypeptide chain elongation
- Every addition of an amino acid to a growing polypeptide chain is the result of an elongation
cycle.
- It composed of three phases: aminoacyl-tRNA binding, the transpeptidation reaction, and
translocation.
- The process is aided by proteins called elongation factors (EF).
- In each turn of the cycle, an amino acid corresponding to the proper mRNA codon is added
to the C-terminal end of the polypeptide chain as the ribosome moves down the mRNA in
the 5’ to 3’ direction.
- At the beginning of an elongation cycle: the P site is filled with either the initiator fMet-tRNA
or a tRNA bearing a growing polypeptide chain (peptidyl-tRNA), and the A and E sites are
empty. Messenger RNA is bound to the ribosome in such a way that the proper codon
interacts with the P site tRNA (e.g., an AUG codon for fMet-tRNA). The next codon is located
within the A site and is ready to accept an aminoacyl-tRNA. In the aminoacyl-tRNA binding
phase, the first phase of the cycle, the aminoacyl-tRNA corresponding to the codon in the A
site is inserted so its anticodon is aligned with the codon on the mRNA. In bacterial cells, this
is aided by two elongation factors and requires the expenditure of one GTP. Once the proper
aminoacyl tRNA is in the A site, the second phase of the elongation cycle, the
transpeptidation reaction, occurs.
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