Unit 3 Flashcards
1
Q
nucleic acid polymer
A
nucleic acid
2
Q
nucleic acid monomer
A
nucleotide
3
Q
nucleotide structure
A
CHONP
- phosphate group
- 5 carbon sugar (pentose. in RNA it is ribose sugar. DNA is deoxyribose sugar. It has the oxygen removed)
- nitrogenous base (called that because it has nitrogen. this is the one thing that varies in nucleotides)
4
Q
DNA Nucleotides
A
- Adenine (2 rings therefore purine)
- cytosine (1 ring therefore pyramidine)
- guanine (2 rings therefore purine)
- thymine (1 ring therefore pyramidine)
5
Q
RNA Nucleotides
A
- Adenine (2 rings therefore purine)
- cytosine (1 ring therefore pyramidine)
- guanine (2 rings therefore purine)
- uracil (1 ring therefore pyramidine)
6
Q
phosphodiester bond
A
O-phosphate – O
7
Q
RNA vs DNA
A
- DNA is more stable
- RNA has a pyramidine base called uracil while DNA has a pyramidine base called thymine
o thymine has better hydrogen bond linkage to create a tighter double stranded structure - RNA is single strand, DNA is double helix
- RNA is found everywhere while DNA can be found only in nucleus
- sugar name: RNA ribose (C#2 OH), DNA deoxyribose (C#2 H)
8
Q
DNA structure
A
A and T form 2 hydrogen bonds
o G and C form 3 hydrogen bonds
- alternates phosphate and ribose. then the nitrogenous base sticks out
- condensation synthesis keep the molecules together so the bases stick out. this creates hydrogen bonding between the two chains which keeps them together
“acid” since the phosphate groups are oriented outwards while the basic parts form the rungs of the ladder. overall, molecule acts acidic.
9
Q
Nitrogenous Base
A
The nitrogenous bases form an alphabet for coding. Words in DNA language are all 3 letters long, this creates 64 possible combinations of nucleotides for 20 amino acids. 3 possible combinates that exist for one same protein. This helps to protect you if your DNA mutates since there is a chance that it makes the same code as was before.
10
Q
hydrogen bonding and protein
A
involved in both secondary and tertiary levels of protein structure
the alpha helices and btal pleated sheets of secondary structure are stabilized by hydrogen bond formation between the amino and carbonyl groups of the amino acid backbone. hydrogen bond formation between r-groups helps stabilize the three dimensional folding of the protein at the tertiary level of structure
11
Q
nucleic acid and hydrogen bonding
A
hydrogen bonds are important for complementary base-pairing between the two strands of nucleic acid that make up an molecule of DNA. complementary base-pairing can also occur within the single nucleic acid strand of an RNA molecule
12
Q
complementary base pairing
A
the A and T (or U), C and T. these bases are complementary in size and this configuration is the most stable hydrogen bonding configuration
13
Q
thymine and adenine
A
have 2 hydrogen bonds
14
Q
cytosine and guanine
A
have 3 hydrogen bonds
15
Q
“Purines always glow”
A
purines are Adenine and Guanine
16
Q
replication
A
when you want to duplicate a cell
- splitting of DNA in half
- then you create another right hand side to match the original left hand split DNA and same thing for right side
- you now have two identical copies of the DNA
17
Q
transcription
A
- how genes turn from just DNA instructions to actual proteins
- how DNA goes to mRNA (messenger RNA)
- first step is the same for replication. you first copy one half of the DNA
- insteaded of Adenine with Thymine, Adenine pairs with Uracil.
- this new mRNA can leave the nucleus, attach to a ribosome and code for a protein
18
Q
translation
A
- how the mRNA turns into an amino acid sequence to turn into a protein
- this sequence from transcription is used. now every three bases codes for a specific amino acid. three bases together are called a codon
- you can have 1 of 4 bases in 3 different places which creates 64 different codons
o this allows you to account for the 20 different amino acids and reduces the danger of mutation - tRNA attaches to amino acids and then matches them to a mRNA to create a sequence of amino acids which create the right protein.
19
Q
uracil vs thymine
A
Uracil is a little less stable than thymine and can make errors more easily. this means that the body would rather have errors in the protein that instructs rather than the instructions themselves because then only some proteins will be wrong, not the instructions itself. Also, mRNA should not be stable because then they would last forever and they are supposed to be messengers.
20
Q
“purines
A
Always Glow”
- 2 rings
21
Q
Replication steps
A
initiation, elongation, termination/completio, post-processing
22
Q
initiation replication
A
In prokaryotic cells like, E. coli there is one single origin of replication (replicon) as the chromosomes are circular. The replication then proceeds until there are two separate chromosomes. In Eukaryotic cells their chromosomes are linear (and much larger - 4.7 million base pairs compared to anywhere from 51-245 million base pairs) and thus have several origins of replication. A large multienzyme complex called a replisome is the machine that carries out DNA systhesis.
First, replication bubbles are opened up by enzyme __. Replication forks are opened further by an enzyme called helices, which uses energy from ATP to unwind the two parent DNA strands. Single stranded binding proteins (SSBs) then attach to the open parts of the helix to keep the parent strands from reattaching. Another enzyme, called DNA gyrase will bind upstream of the replication fork to reduce torsional strain caused by the unwinding strands. This enzyme is an example of a topoisomerase, in which Eukaryotic cells use several types to reduce torsion.
Once the DNA is unwound, the enzyme DNA polymerase will be able to replicate the DNA strand. However, this enzyme can only assemble DNA if there is something to add to. The start of the sequence will be initiated by the enzyme DNA primase, which will create a short sequence (about 16 bases) of RNA that DNA polymerase can add to.
23
Q
elongation replication
A
- Deoxyribonucleoside triphosphates hydrogen bond to the exposed bases on the single stranded DNA according to the base pairing rules, with an accuracy of about 99.99%
- A collection of enzymes [called a replisomes] containing DNA Polymerase III removes two phosphates from the 5’ end of the nucleotide and uses the energy to bind it to the 3’ carbon of the previous nucleotide. The new DNA chains thus grow in the 5’ to 3’ direction.[The DNA polymerases in Eukaryotes are called Pol δ and Pol ε. Their role is similar to Pol III..]
- At each replication fork, one new chain can grow continuously from a single RNA primer, as the fork is extending in its 5’ to 3’ direction. - This is called the leading strand.
- For the other new chain, however, the fork is extending in the 3’ to 5’ direction. Numerous primers must be started, and this chain must grow in short sections that elongate back toward the origin. These short sections of DNA, called Okazaki fragments, are typically a few thousand bases long.
- When adjacent replication forks meet, the entire DNA molecule has been copied, but each new strand has alternating long continuous stretches (leading strands) and stretches of short Okazaki fragments (lagging strands). Each leading strand and each Okazaki fragment has a short stretch of RNA where it started.
A multi subunit protein complex called the DNA Pol III remains attached to the template strand and continually synthesizes DNA. It is held in place by a subunit of the protein called the β-subunit, β-clamp or sliding clamp.
24
Q
termination/completion replication
A
- An enzyme called DNA Polymerase I (an exonuclease) begins removing RNA nucleotides from one section and adding the appropriate DNA nucleotides to the end of the adjacent strand.
- All the RNA nucleotides are replaced, but the last nucleotide cannot be bonded by polymerase because it no longer has a triphosphate to provide the required energy. Another enzyme, called DNA ligase, finally links all the fragments together.
- Now each daughter strand of DNA is a complete complement for its parent strand.
When replication is complete, DNA gyrase will separate the two chromosomes.
25
Post-processing replication
- Pol III (or Pol ε and Pol δ) in the replisome proofreads the new DNA as it adds bases, checking for appropriate width of the helix. If it finds mistakes, it backs up, removes the newly added bases, and replaces them with bases that properly complement the parent strand.
- Many other proofing enzymes check DNA constantly and attempt to repair any damage done by radiation, free radicals or other mistakes. These include:
Mismatch repair enzymes that detect shape irregularities in the double helix and replace mismatched bases with complementary pairs.
- Because eukaryotic chromosomes are linear, there is always a little section on each lagging strand that cannot be replicated, because it lies beyond the last RNA priming site. Thus, eukaryotic chromosomes tend to become slightly shorter at each division. This slow erosion of the chromosomes may be a major factor in cell and tissue aging. Single celled eukaryotes and the germ cells of multicellular ones produce an enzyme called telomerase.
- Telomerase contains a short stretch of RNA, which it uses as a template to add multiple repeats of DNA to the end of the chromosome. Once telomerase is shut down in specialized cells, the telomers shorten with each division. The initial length of these “telomeres” may determine the number of divisions a cell can make before it dies. Cells with active telomerase are essentially immortal.
26
lagging strand
has the okazaki fragments
27
why 5' to 3'
It builds in a 5’ to 3’ direction, because DNA Pol. III can only synthesize DNA in the 5’ to 3’ direction. Cells build this way, because the energy for the synthesis is provided by deoxynucleoside triphosphates. If it were in the other direction, the energy would have to come from the 5’ end of the growing molecule.
28
During what two natural cell processes would DNA replication occur?(1)
Mitosis and Meiosis
29
Why is the structure of the DNA double helix important to replication?
It allows two copies to be produced at once.
30
RNA transcription steps
initiation, elongation, termination, post-processing
31
initiation (RNA transcription)
- Each gene has a promoter region “upstream” that must be activated to start transcription. Promoters can be turned on or off by signals from the cell. [Bacteria have many of their genes arranged in operons that contain the genes for several related enzymes. An operon requires only one promoter.]
- A transcription factor (usually protein) must first bind to the promoter before the gene can be read. [In many eukaryotic genes, the transcription factor binds to a region called a TATA box, a stretch of DNA containing the sequence TATAAA, approximately 25 bases upstream from the area to be transcribed. In bacteria, the area is usually rich in T and A, but less uniform.]
- Other transcription factors and enzymes then bind to the activated promoter to form an RNA Polymerase holoenzyme. These additional transcription factors allow the cell control over which genes are active at any time.
32
elongation (RNA transcription)
As transcription begins, the enzyme will change shape, leave the
promoter region and then proceed along the template strand. The
enzyme covers a large span of the DNA, but only unwinds about 10
bases at a time. This is called the transcription bubble. It then synthesizes a single stranded RNA molecule that is complementary
to the template strand in a 5' to 3' direction. As the polymerase
passes, the DNA is rewound.
33
Termination (RNA transcription)
DNA transcription will complete when the RNA polymerase reaches a
specific nucleotide sequence in the DNA. This will cause the enzyme to
detach from the DNA strand. As the mRNA is released from the DNA,
the DNA double helix is reformed.
This sequence usually has many guanine and cytosine pairs and is
followed by a sequence of adenine and thymine pairs. The GC pairs
twist and hydrogen bond, forming a hairpin. At this point the RNA
polymerase has bonded a series of uracil bases to the adenine in the
template strand. This puts pressure on the weak intermolecular forces
between U and A and the RNA dissociates from the strand.
Termination in eukaryotic cells is recognized by a specific
sequence called a polyadenylation sequence which codes for
AAUAAA. About 10-35 nucleotides downstream from this, the
RNA Pol II dissociates from the DNA.
34
post processing (RNA transcription)
prokaryotic is ready to be translated right
after it is transcribed. It will bind to ribosomes straight away (even
sometimes before transcription is complete) and the protein will be
produced.
- In eukaryotes, the mRNA must be processed before it can leave the
nucleus and produce a protein. The mRNA that is produced by RNA
Pol II is referred to as the primary transcript or precursor mRNA
(pre-mRNA). The processed mRNA is called mature mRNA. The processing that occurs includes:
Addition of a 5' cap - this consists of a methylated GTP molecule
that is attached to the 5' end of the transcript via the phosphate (5'-
5' bond). The purpose of this protects the RNA and helps it to bind
to a ribosome. makes it resistant to degrading enzymes in the cytoplasm.
Addition of a 3' poly-A tail - an enzyme adds a series of adenine
nucleotides, which makes the mRNA more stable and allows it to
exist longer in the cytoplasm. The length of poly-A tail determines how long the mRNA will be active before it becomes degraded.
RNA splicing - eukaryotic genes contain non-coding regions called
introns, which are interspersed inbetween coding regions called
exons.
35
How does RNA differ from DNA (three major differences)
single stranded
ribose sugar
uracil instead of thymine
36
How did the DNA determine the structure of the mRNA?
The mRNA is produced in the 5’ to 3’ direction and is written complementary to the template strand.
37
Why is RNA required as a messenger? Why can’t the DNA simply carry information where it is needed? Think of as many advantages of using mRNA as you can.
mRNA is produced from DNA, because there is only one DNA molecule. If this molecule is damaged or digested, it could result in the death of the cell. DNA is also a much longer molecule, which would be difficult to transport and attach to the site of protein synthesis. Another advantage of mRNA is that it can remain active producing protein as long as required by the cell. This would be more efficient than if you had to use energy to unwind the DNA every time you need to produce a polypeptide.
38
How would the RNA transcribed in a real cell differ from your simple string of codons. How would it have to be changed before translation if this was a real Eukaryotic cell?
It would have a methylated GTP 5’ cap and it would have a poly-A tail at the 3’ end. It would need to have had the introns spliced out before translation.
39
translation
Once the mRNA transcript is produced, it is then translated in to a
protein the second stage of the central dogma. In this process,
codons are read to produce the primary confirmation of a
polypeptide. The interpretation of codons is done by transfer RNA or tRNA. A tRNA molecule is a single stranded RNA molecule, which is folded into a characteristic clover-leaf shape. The stems of
the looped areas are held together by intramolecular base pairing.
There are two funcitonal regions of the tRNA molecule. One
contains the anticodon loop, which is a stretch of nucleotides that is complimentary to the mRNA codon. The other is at the 3' single-
stranded end of the molecule, where the amino acid is attached and
is called the acceptor end.
40
the charging reaction
The enzyme that attaches the appropriate amino acid to the
corresponding tRNA molecule is called aminoacyl-tRNA
synthetase. There are 45 enzymes and 45 tRNAs that carry the 20 amino acids. The amino acid is added to the tRNA using the
energy from ATP. This produces a charged tRNA, which will not
require anymore energy when being added to the growing
polypeptide chain.
41
ribosomes in translation
All three steps of translation take place on the ribosome, which is a large
molecule made of two subunits that consist of protein and rRNA
(ribosomal RNA). The ribosome will bind two charged tRNA molecules so
that a peptide bond can be formed between the amino acid units.
Ribosomes have 3 binding sites:
P site (peptidyl) - binds the tRNA that attaches to the growing polypeptide
chain.
A site (aminoacyl) - binds the tRNA carrying the next amino acid.
E site (exit) - binds the tRNA that carried the previous amino acid.
tRNA molecules will proceed during elongation from site A to P to E.
42
initiation translation
In both prokaryotic cells and eukaryotic cells, initiation involves a
series of proteins called initiation factors. The exact mechanism
and types of proteins are different, but the key aspects of the
process are similar.
rRNA in the small subunit of the ribosome will bind to the mRNA,
near the start codon (AUG on the mRNA) along with the initiator
tRNA-met (carries methionine and has an anticodon UAC). The
large subunit of the ribosome is then added, positioning the initiator
tRNA at the P site.
43
elongation translation
The elongation of a polypeptide chain starts with the binding of a new
tRNA to the A site on the ribosome. Once bonded to the A site, the
ribosome catalyzes a reaction that forms a peptide bond between the
open amino end of the amino acid in the A site and carboxyl end of
the amino acid in the P site. The mRNA then moves one codon
forward so that the tRNA with the growing polypeptide chain is now in
the P site. The 'empty' tRNA is now in the E (exit) site and the A site
is open, ready for the next charged tRNA to bind. This process is
called translocation. The tRNA in the E site leaves and another tRNA binds repeating the process and adding another amino acid to
the polypeptide chain.
44
termination translation
Elongation will continue until one of the stop codons is
reached. These codons do not bind tRNA, but instead bind
proteins called release factors or termination factors. Binding of one of these factors to the A site, causes an enzyme to
break the bond between the polypeptide chain and the tRNA in
the P site. The ribosomal complex then dissociates from the
mRNA, which will remain around to be translated again until it
is degraded by ribonuclease enzymes.
45
RNA polymerase
RNA polemerase (prokaryotic) is quaternary structure
- help the enzyme bind to the DNA
- must face in a way that it can build in a way of 5’ to 3’
- there are promoters that are always 10 bases and 35 bases upstream from the start of the gene
- the unique 3d shape of promoter regions helps enzyme bind
- the promoter region helps the enzyme bind facing the right way and help identify the gene that it is working with
- the -10 always has an alternating adenine and thymine pattern (TATA box) must be a TATA pattern!
46
eukaryotic transcription
- only one TATA box promoter
| - it faces the right way because it binds to the transcription factors which make sure that it is facing 5’ to 3’
47
termination transcription prokaryotic
o to recognize when it stops, you develop the hairpin loop
o there is an alternation sequence of guanine and cytosines, the loop isn’t that sequence, then it follows with more guanine and cytoseine
• formation happens because the molecule bends around and the bases will line up and hydrogen bond with each other forming a hairpin loop
- formation of hairpin causes the polymerase enzyme to pause as the hairpin gets caught in the enzyme
- when it gets stuck, the mRNA sequence in the enzyme is a series of uracil-adenine pars which is weak hydrogen bonding which separates the mRNA molecule from the DNA molecule
o this also makes the enzyme dissociate from the DNA
48
termination transcription eukaryotic
o when it reaches the sequence AAUAA which signals the enzyme to stop and break off
49
post processing transcription prokaryotes
- prokaryotic cells don't need to further process their mRNA
o that is because we have an nucleus while everything is happening in the cytoplasm
o they overlap the process of translation
o they start translation before transcription even finishes
o ribosomes do this transcription
o sometimes there is more than one ribosome. instead of one gene coding for one protein, multiple genes can be produced by one mRNA and produce like three polypeptides at the same time
50
post processing eukaryotes
o the mRNA is actually only the primary transcript and is not able to leave the nucleus and produce a protein sicne there are things that are used to digest nucleic acids outside
• 3 things.
• there is a 5’ cap which is made of GTP (methalated)
• enzyme won’t recognize it with its active site
• Poly-A tail does not stop enzymes from digesting the 3’ end but the length of the AAAAA pattern determines how long it will last in the cytoplasm. longer= more protein made
o this regulates how much and when
o there is an enzyme that can change the length of the AAAA
• mRNA must be spliced
o code for protein is called an exon
o code for nothing is an intron (doesn’t code for protein that you are trying to make)
o we have to cut out the introns and leave in the exons
o snRNA recognizes the exon and intron space
• it is in a protein which is called snRNP’s
o they cut at the edges of the introns and exons and glue together the exons
o the four snRPS’s are called a slpiceosome
51
telomeres
Eukaryotic chromosomes are linear and so eventually replication
cease at the end of the chromosome. These specialized structures
are called telomeres and protect the ends of chromosomes from the
activity of nuclease enzymes. Telomeres do not code for anything
and are actually just repeats of the sequence 5' TTGGGG 3'. A
human telomere can consist of up to 2000 of these sequences. This
linear nature and the action of DNA polymerases causes a unique
problem when the replication fork reaches the end of a
chromosome. Eventually, the lagging strand runs out of room to
place a new RNA primer. When the last primer is removed, DNA Pol
I is unable to replace the segment. After the next replication, the
chromosome is slightly shorter.
This shortening gives most cells a finite life span (about 100
replications). This is not true for all cells. Single-celled organisms,
germ cells, stem cells and cancer cells all have the ability to maintain
their telomeres using an enzyme called telomerase.
52
telomerase
Using an RNA template, telomerase will lengthen the
overhanging single stranded portion of the chromosome. Once
this is lengthened, DNA primase can add a RNA primer to the
shorter sequence and then DNA Pol III can sequence an
Okazaki fragment. This lengthens the repeating segment,
ensuring that no coded DNA is eliminated.
53
gene expression
Gene expression is a term that refers to the process by which DNA directs the synthesis of proteins.
DNA to RNA to Protein
The DNA to RNA part happens in a process called transcription, while the RNA to protein part is called translation.
54
DNA to RNA
DNA is double stranded and RNA is single stranded. This means that only
one strand of the DNA is going to be copied. We call the strand that will be
copied the coding strand. The strand that is not copied is the template strand
(because the RNA will be complementary to it).
The RNA that will be produced will be a copy of the coding strand, but will
have Uracil instead of Thymine bases. The RNA strand is then read in groups of three nucleotides called codons.
55
different RNA polymerase's (don't need to know all)
RNA polymerase I - transcribes rRNA (ribosomal RNA)
RNA polymerase II - transcribes mRNA and snRNA (small nuclear RNA)
RNA polymerase III - transcribes tRNA (transfer RNA) and other small RNA
eukaryotic cells have only one polymerase
56
pre-mRNA splicing
The borders of introns and exons are recognized by small nuclear
ribonucleic particles or snRNP. These are complexes which are comprised of protein and small nuclear RNA
(snRNA). Once bound, they cluster together to form a spliceosome.
The spliceosome then removes the 5' end of the intron and bonds it
with a specific point on the intron called a branch site. The 3' end is
then cut and the ends of the exon are joined together. The splicosome
then dissociates.
57
polymerases
Can only synthesize in one direction
58
mutation
ny change to
the heritable material. It may involve a small change in the
internal code of DNA, or a gross change to a chromosome
59
mutagen
A
substance that causes a mutation or increases the number of
mutations above the typical levels
60
point mutation
change in one base code in the DNA. They may
result from copying errors ("typos") or from external damage to the
DNA.
can:
- have no effect (same amino acid different codon)
- alter a single amino acid
- alter reading frame (insertion or deletion)
- create a start codon (incorrect ribosome alignment) or destroy start (no gene expression) or end codon (extra amino acid addition)
- alter regulatory regions (change in activation level or timing)
61
translocation mutation
occurs when a stretch of DNA code is
removed or added at a new location. Transposable elements in DNA
may move around, and viruses may insert DNA where it doesn't
belong. DNA copying mechanisms may sometimes "skip" sections
of DNA. whole genes are moved
can
- add extra aminos
- create some incorrect aminos in protein
- separate gene and its regulatory region
- move an active regulatory region to an inactive portion of DNA
- add additional start/stop codons
62
sense mutation
type of point mutation that makes sense even with letter changed (the codon still has the same protein bc codons repeat)
63
missense mutation
1) type of point mutation that changes the codon but is still possible to exist. it usually has minimal phenotypic effect since it is just one codon changed (substitution)
2) type of point mutation that changes the codon to something that doesn’t make sense. this has a major phenotypic effect since the codon isn’t very possible to exist (substitution)
64
nonsense mutation
the substitution causes early termination of the polypeptide chain (one word is different and now it is the end codon) which causes misfolding
65
frameshift mutation
1) when one base is deleted then all the other ones shift (deletion) which creates a missense mutation
2) when one base is added the all the other ones shift (insertion) which creates a missense mutation
protein is completely changed which affects tertiary folding but NOT ALWAYS (Codon can still be read maybe)
66
chromosomal mutation
LARGEST EFFECT
- causes Inversion of one or multiple codons which makes it loose meaning
- causes a Tandem repeat (codons are repeated) which changes the meaning
67
DNA pol III
non specific repair
DNA pol III (writes the DNA strand) checked the strand for consistency as it goes. if it finds a mismatch, it goes backwards, removing the new strand (like an exonuclease) and writes it again. this creates a 1 in 107 error.
68
excision repair
non specific repair
they can’t find which strand has the mutation since it is not writing it. it comes after both DNA is written. Parent strands of DNA has methyl groups attached which is a way to regulate gene expression. When a gene has a methyl group, it is like an off switch.
i. this protein has three parts. One binds to the methyl group, one binds to the mistake, one binds in between to allow for change in structure. this then cuts from the bases in between which is like an endonuclease.
ii. then DNA polymerase rewrites the strand and ligase fills the gap
iii. enzyme will come and methylate the daughter strand and correction is complete
69
photolyase
specific repair
a. photolyase uses light energy to break bonds but only targets one mutation. UV radiation can cause two Thymine letters to bond together to create a thymine dimer which is the leading cause to skin cancer.
i. photolyase uses wavelengths of blue light to break the bond and correct the error. OR it could also do excision repair using excision repair enzymes (DNA polymerase III, ligase etc.)
thymine dimer can cause other mutations or negatively effect replication
70
why might DNA be removed
An endonuclease (excision repair enzyme) has removed a mutated stretch of DNA to repair it, perhaps because UV light caused a thymine dimer at THE REMOVED DNA
71
enzymes to replace DNA?
ligase and DNA polymerase III
72
where would transcription factor bind if eukaryotic?
TATA box
73
coding strand?
the strand with the TATA box
74
where would transcription begin on strand
25 bases downstream from TATA box
75
molecules to activate transcription
Transcription factors and RNA polymerase enzyme (Pol II in eukaryotic cells)
76
enzyme required for transcription?
RNA polymerase II
77
where would transcription
end of sequence AAUAAA
78
RNA processing if it was a eukaryote?
this RNA is pre-mRNA. must add Methylated GTP cap (methyl-GTP attached by a 5'-5' bond) and ploy-A tail added, introns spliced out
79
where would RNA transcribed begin translation?
at start codon AUG
80
What cellular and chemical components would be necessary for translation to begin?
large and small ribosomal subunits, met-tRNA, GTP, mRNA
81
What is the role of aminoacyl-tRNA-synthetase enzymes in translation?
Bind each tRNA with it’s appropriate amino acid in the cytoplasm (There are 45 tRNA’s and 45 synthetases)
82
DNA barcoding
it is taking samples of DNA and processing them to get a barcode which has four unique states, over 600 positions
ethics:
83
restriction endonucleases
Enzymes called restriction endonucleases (REs), derived from bacteria
Cut DNA at particular base sequences in the middle of the double helix
Give molecular geneticists a set of tools that allow them to cut any DNA at very specific points.
the cut sites are offset from another and can be merged with ANY dna that was cut from the same endonuclease
Ethics:
84
gel electropheresis
separates molecules based on size relies on fact that DNA has a negative charge
1) make an electrophoresis device with wells to place DNA on the -ve side and the other end with +ve charge. the gel (agarose) allows DNA to pass through it and since DNA is negatively charged, it is going to travel to the positive side.
2) most times, the DNA is cut using restriction endonuclease which cuts the DNA at the same identification points. since the DNA copies are unique, those pieces will be different sizes
3) shorter pieces will travel farther to the +ve end while longer pieces will stay close to the well
4) DNA bands will be placed in dyes and those samples would be analyzed
5) compare the DNA and the one with the most similar bars would be the match for that DNA
6) you can use a DNA ladder with base pairs marked on it (can determine the length of the DNA based on how far it travelled)`
85
PCR (polymerase chain reaction)
1) take DNA samples and to denature the strands (separate them), you apply heat. problem is that enzymes would denature at that heat so they won't be able to replicate. solution is a bacteria that can make DNA at high temp called TAQ polymerase. it is applied to copy the dNA. you keep doing this and its exponential growth
- you use this to get samples for gel electrophoresis
86
DNA sequencing (??)
DNA is purified, then cells and nuclei are broken open. Large pieces of DNA such as whole chromosomes or genomes are cut into smaller pieces. these pieces are placed in bacteria (since the DNA can reproduce), this bacteria is placed in a culture and they multiply a lot. This way, the DNA can be multiplied and is now a clone.
87
What is the goal of PCR?
The goal is to produce multiple copies of a small segment of DNA, which is needed for research.
88
What reactants and enzyme do you have to add to the tube? PCR
Extracted DNA, primers, nucleotides, DNA Polymerase enzyme.
89
Why are two primers necessary to isolate a section of DNA? PCR
one primer attaches to the top strand at one end of your segment of interest, and the other primer attaches to the bottom strand at the other end. In most cases, 2 primers that are 20 or so nucleotides long will target just one place in the entire genome.
Primers are also necessary because DNA polymerase can't attach at just any old place and start copying away. It can only add onto an existing piece of DNA.
90
What temperatures does the PCR need to be run at and what is the purpose of each temperature?
95oC – separate DNA into single strands
50oC – cooler temperature for primers to anneal to the single strands of DNA
72 oC – to activate DNA polymerase enzyme
91
After how many cycles will you finally get your DNA of interest? PCR
3
92
Brainstorm at least three situation in which you might want to amplify a tiny sample of DNA
Identify bacteria, diagnose disease, criminal forensics, tissue typing for organ transplantation, genetic testing, paternity testing, DNA sequencing, phylogenetics
93
What is the goal of sequencing?
The goal is to know the DNA sequence for an organism. \
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Briefly, how are clone libraries of DNA made?
DNA is removed from cells by precipitation. The DNA is cut up and inserted into vectors, which are placed in bacteria to be reproduced.
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Why do you need millions of copies of the DNA to sequence it?
As you stop replication, you will be able to identify the base, but you will need multiple copies to identify all of the bases.
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What enzyme is required for the sequencing reaction
DNA polymerase
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What is the function of the labelled dideoxynucleotide triphosphates (ddNTPs)
It stops the replication of DNA at that base, because it does not have a 3’ OH group to attach to. They are labelled so they can be detected and identified.
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In your own words, explain what will happen as DNA polymerase extends the millions of new complementary DNA strands.
It will add the bases complementary to the template strand of DNA. Eventually, one of the small amounts of the dideoxynucleotides will bind and stop replication
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Since only sequences of thousands of bases can be sequenced at a time, but genomes can be between 3 million and 3 billion base pairs long, how are the fragments of sequenced DNA combined?
Using capillary electrophoresis, the different length segments are separated. They pass through a laser in order of size and the fluorescent ddNTP will have light transmit through it at a certain wavelength. This can be detected by a photocell attached to a computer. The computer displays it as a trace which can interpret the wavelength of light as the appropriate base.
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regulation in transcription
- since we are made of more than one cell, we need some mechanism to regulate which cells make what and how much
- one way to do that is to condense the DNA sicne when it is condensed, it is inaccessible
- largest area of regulation is the promoter regions
o there are specific transcription factors
o sometimes the bind to the enzyme to block it, not allowing transcription to take place
o sometimes, they allow more transcription so more protein to be made
o this is not in bacteria and prokaryotic since they only have one cell and don’t have to differentiate
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Lac Operon (prokaryotic cells)
An operon, or group of genes with a single promoter (transcribed as a single mRNA). In the Lac Operon there are 3 genes required to bring in lactose and then digest it to release galactose and glucose. This operon is normally switched off. It is controlled by a separate repressor protein that will be bound to a region between the promoter and the start site called the operator. This blocks transcription. When glucose is not present in the cell an activator protein is switched on that will promote transcription. When small amounts of lactose are available in the cell they will alter the shape of the repressor protein removing it from the DNA and allowing transcription.
This is and example that impacts the type of protein in the cell.
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Trp Operon (prokaryotic cells)
An operon, or group of genes with a single promoter (transcribed as a single mRNA). In the Lac Operon there are 3 genes required to bring in lactose and then digest it to release galactose and glucose. This operon is normally switched off. It is controlled by a separate repressor protein that will be bound to a region between the promoter and the start site called the operator. This blocks transcription. When glucose is not present in the cell an activator protein is switched on that will promote transcription. When small amounts of lactose are available in the cell they will alter the shape of the repressor protein removing it from the DNA and allowing transcription.
This is and example that impacts the type of protein in the cell.
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transcription factors (enhancers and activators)
There are general transcription factors that are all required for transcription, but there are also specific transcription factors that can increase or decrease transcription. Activators bind to enhancer regions on the DNA to increase transcription. Repressors bind to silencer regions and decrease transcription. This will affect the amount of a protein that is made.
104
DNA methylation and histone modification
regulates: pre transcription
Methyl groups can be added to DNA not changing the structure of code but can prevent the binding of RNA polymerase. This inhibits transcription.
Histones are proteins that contribute to DNA folding into chromosomes. The more folded the chromatin is the less accessible the DNA is. The more relaxed the chromatin is, the more available it is for transcription.
These affect the type of protein.
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Alternative Splicing
regulates: between transcription and translation
Introns are sections of the RNA that are removed because they don’t code for protein. Exons are sections that code for protein and are linked or together to make the final mRNA. Different portions of an mRNA can be selected for use as exons. This allows either of two (or more) mRNA molecules to be made from one pre-mRNA
This affects the type of protein..
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Small RNA (RNA interference)
regulates: transcription
Small RNA molecules called miRNA can bind to specific sequences of mRNA. They then recruit an enzyme which breaks down the RNA reducing the amount of time it is available for translation.
This will affect the amount of a protein.
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Regulation of Translation
regulates: after transcription
Many helper proteins are required for translation. These proteins can be regulated as well.
This will affect the amount of protein (or timing).
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Phosphorylation
regulates: after transcription
Phosphorylation can activate or deactivate proteins. This can regulate the timing of protein activation or the amount that is active.
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Protein degradation (ubiquitin)
regulates: after transcription
Proteins are tagged for degradation by the addition of a chemical marker, called ubiquitin. Ubiquitin-tagged proteins are taken to the proteasome and broken down into amino acids.
Will control the amount of protein.
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what components does replication require?
DNA polymerase, a template strand and nucleotides
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where origins of replication?
opened by replisome. origins of replication are generally rich in A-T pairs, which are more weakly hydrogen-bonded than G-C pairs.
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DNA pol I
is an exonuclease
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Where does transcription start (eukaryotes, prokaryotes)
In prokaryotes, they have two promoters for a gene, one which is 35
and one that is 10 nucleotides upstream from the start of
transcription. These two promoters are unique and indicate the site
of initiation as well as what direction the enzyme should transcribe.
In eukaryotic cells, the core promoter site is approximately 25
nucleotides upstream of the starting site and is rich in adenine and
thymine. This is why it is referred to as the TATA box. This is similar to the one in prokaryotic cells that is 10 nucleotides
upstream.
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transcription termination difference in (eukaryotes, prokaryotes)
prokaryotes: this point the RNA
polymerase has bonded a series of uracil bases to the adenine in the
template strand. This puts pressure on the weak intermolecular forces
between U and A and the RNA dissociates from the strand.
eukaryotes: Termination in eukaryotic cells is recognized by a specific
sequence called a polyadenylation sequence which codes for
AAUAAA. About 10-35 nucleotides downstream from this, the
RNA Pol II dissociates from the DNA. q
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snRNP
Made of protein and snRNA
116
even leading strand
has RNA primer but no fragments after that
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ATP is required for
start of transcription and charging reaction of translation
118
DNA pol vs RNA pol
DNA pol works 3'5' not RNA pol
119
downstream is
to the right
120
replication end?
replication ends at forks meeting
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transcription eukaryotes and prokaryotes
- prokaryotic has two promoters
- eukaryotic ends on AAuAAAA
- prokaryotic ends at hairpin loop at gc pairs (h bond) and fall off at UT pairs
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GTP?
GTP is attached to 5' cap and this energy is used to bind small ribosomal rRNA
translation start at AUG codon but is recognized at tRNA at 5' cap
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eukaryote translation location
- translation happens in the cytoplasm!
