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1

adult stem cell

Adult Stem Cells (or Tissue-specific Stem Cells)

Adult stem cells are tissue-specific, meaning they are found in a given tissue in our bodies and generate the mature cell types within that particular tissue or organ. The term ‘adult stem cells’ is often used very broadly and may include fetal and cord blood stem cells.

Example: new erythrocytes (red blood cells) are generated from adult blood-forming stem cells in bone marrow

2

Fetal Stem Cells

Fetal stem cells are taken from the fetus. The developing baby is referred to as a fetus from approximately 10 weeks of gestation.

Most tissues in a fetus contain stem cells that drive the rapid growth and development of the organs. Like adult stem cells, fetal stem cells are generally tissue- specific, and generate the mature cell types within the particular tissue or organ in which they are found. 

3

Cord Blood Stem Cells 

Taken from blood in the umbilical cord, rich in blood-forming stem cells. The applications of cord blood are similar to those of adult bone marrow and are currently used to treat diseases and conditions of the blood or to restore the blood system after treatment for specific cancers. Like the stem cells in adult bone marrow, cord blood stem cells are tissue-specific. 

4

Embryonic Stem Cells

Embryonic stem cells are derived from very early embryos and can in theory give rise to all cell types in the body. However, coaxing these cells to become a particular cell type in the laboratory is not trivial. Furthermore, embryonic stem cells carry the risk of transforming into cancerous tissue after transplantation.

To be used in cell transplant treatments the cells will most likely need to be directed into a more mature cell type, both to be therapeutically effective and to minimize risk that cancers develop. While these cells are already helping us better understand diseases and hold enormous promise for future therapies, there are currently no treatments using embryonic stem cells accepted by the medical community. 

5

iPS cells

Induced Pluripotent Stem Cells (iPS cells)

In 2006, scientists discovered how to “reprogram” cells with a specialized function (for example, skin cells) in the laboratory, so that they behave like an embryonic stem cell. These cells, called induced pluripotent cells or iPS cells, are created by inducing the specialized cells to express genes that are normally made in embryonic stem cells and that control how the cell functions. Embryonic stem cells and iPS cells share many characteristics, including the ability become the cells of all organs and tissues, but they are not identical and can sometimes behave slightly differently. IPS cells are a powerful method for creating patient- and disease-specific cell lines for research. However, the techniques used to make them need to be carefully refined before they can be used to generate iPS cells suitable for safe and effective therapies. 

6

Difference Between Adenine and Adenosine, AMP

Because of their technical nature and close relation, many people use the adenine and adenosine interchangeably like in the case of AMP or adenosine monophosphate wherein people substitute it with the word adenine making it adenine monophosphate, which is an incorrectly notated molecular name.

Adenine is a nucleobase (a purine derivative) while the adenosine is a nucleotide. These nucleotides are glycosylamines that has a certain nucleobase attached to them.

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7

The 70S ribosome is prokaryotic or eukaryotic? Composed of what subunits? Those subunits are composed of what DNA and how many proteins?

Prokaryotic, the 70S ribosome is made of the 50S (5S, 23S rRNA and 34 proteins) and the 30S (16S rRNA and 21 proteins)

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The 80S ribosome is prokaryotic or eukaryotic? Composed of what subunits? Those subunits are composed of what DNA and how many proteins?

Eukaryotic, the 80S ribosome is made of the 60S (5S, 5.8S, 28S rRNA and ~49 proteins) and the 40S (18S rRNA and ~33 proteins)

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What end does the small ribosomal subunit bind to in prokaryotic mRNA?

Near the 5' end, based on complementarity  between the 16S rRNA and a conserved sequence found in the 5’ untranslated region called the Shine-Delgarno sequence

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Shine-Delgarno sequence

In prokaryotes (such as E. coli) the RBS typically lies about 7 nucleotides upstream from the start codon (i.e., the first AUG). The sequence itself in general is called the "Shine-Dalgarno" sequence after its discoverers, regardless of the exact identity of the bases. Strong Shine-Dalgarno sequences are rich in purines (A's,G's), and the "Shine-Dalgarno consensus" sequence -- derived statistically from lining up many well-characterized strong ribosome binding sites -- has the sequence AGGAGG. The complementary sequence (CCUCCU) occurs at the 3'-end of the structural RNA ("16S") of the small ribosomal subunit (30S) and it base-pairs with the Shine-Dalgarno sequence in the mRNA to facilitate proper initiation of protein synthesis.

Protein synthesis in eukaryotes differs from this model. The 5' end of the mRNA has a modified chemical structure ("cap") recognized by the ribosome, which then binds the mRNA and moves along it ("scans") until it finds the first AUG codon. A characteristic pattern of bases (called a "Kozak sequence") is sometimes found around that codon and assists in positioning the mRNA correctly in a manner reminiscent of the Shine-Dalgarno sequence, but not involving base pairing with the ribosomal RNA.

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After locating the Shine-Delgarno sequence, what happens next?

The small subunit (30S) then scans along until it encounters the first AUG (initiation) codon. This establishes the reading frame and the rest of the mRNA is read in triplets following the AUG.

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12

AUG codes for what amino acid in eukaryotes? prokaryotes?

The start codon (AUG) always codes for methionine in eukaryotes and a modified Met (fMet) in prokaryotes

13

What modifications are required in eukaroytic mRNA to initiate translation?

Communication  between  the  5' cap  and  the  poly(A)  tail  takes  place  through  the  translation  factor  eIF-4G  (eukaryotic  initiation factor),  which  is required  for  translation.

14

What are the three ribosome binding sites?

E-site (exit, the site where old tRNAs exit the ribosome)

P-site (peptidyl, the site with the growing polypeptide chain)

A-site (aminoacyl, the site with the newly-arrived tRNA)

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tRNA

tRNA is an adaptor moleculecomposed of RNA, typically 76 to 90 nucleotides in length, that serves as the physical link between the mRNA and the amino acid sequence of proteins. It does this by carrying an amino acid to the protein synthetic machinery of a cell (ribosome) as directed by a three-nucleotide sequence (codon) in a messenger RNA (mRNA). As such, tRNAs are a necessary component of translation, the biological synthesis of new proteins according to the genetic code.

16

In eukaryotes, where does the small ribosomal subunit bind to the mRNA?

The  small  ribosomal  subunit  binds  to  the  5' cap  with the help of eIFs (eukaryotic initiation factors, forming the 40S initiation complex) and  scans  for  the  first  AUG  codon. 

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17

What does the initiator tRNA use for energy when it is bound to mRNA?

It hydrolyses GTP (guanosine triphosphate), which also has the role of a source of energy or an activator of substrates in metabolic reactions, like that of ATP, but more specific. It is used as a source of energy for protein synthesis and gluconeogenesis.

18

What happens to the small ribosomal subunit after eIF-2 and other initiation factors dissociate?

The large ribosomal subunit binds, and elongation begins

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Describe the three-step elongation cycle

The three step elongation cycle:

Step 1: an aminoacyl-tRNA binds to the vacant A site

Step 2: a new peptide bond is formed

Step 3: The spend tRNA is ejected and the ribosome is "reset" so that the next aminoacyl-tRNA can bind

20

What binds stop codons?

Instead of tRNAs, which bind all other codons, stop codons (UAG, UAA, UGA) have no corresponding tRNAs and are instead bound by proteins known as release factors, which terminate elongation and eject the completed polypeptide

21

How many stop codons are there?

Three, UAG, UAA, UGA

22

Describe the accuracy of the translation process, what trade-off does this require?

Translation is extremely accurate, and so requires large amounts of energy, more than any other biosynthetic process.

Steps include making sure the mRNA is complete and intact before translation, proofreading the aminoacylation of tRNA, proofreading codon/anticodon matches, etc.

23

What is the mechanism of peptidyl transferase?

Adenine 2451 of the large rRNA catalyses the reaction. It withdraws a proton from the α-amino group of the residue in the A-site, allowing the N to attack the carboxyl group of the nascent protein in the P-site.  The protonated A2451 donates the H to the P-site tRNA, releasing the tRNA from the nascent protein and returning adenine to its original chemical state.

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24

How do antibodies bind antigens?

Lock-and-key fit, which is very selective

25

How many different foriegn molecules can the human body respond against?

106

26

How many kinds of antibodies does one B cell make?

One antibody that recognises one antigen. If we can recognise 106 different antibodies, then we need 106 different B cells.

27

If we have so many types of B cells, how can we accomodate them all?

B cells arrest their development as ‘small resting B cells’

–  When a small resting B cell contacts the specific antigen it recognizes, it binds to the antigen and begins dividing rapidly, forming a clone of identical B cells. Only multiplies when it recognises its antigen

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28

What do B cells mature into?

Plasma cells that secrete antibodies

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How many molecules of antibodies can plasma cells produce per hour?

10 million per hour

30

Describe antibodies

Y-shaped structure consisting of 2 sets of 2 identical chains, light and heavy.

The Fab fragment is the portion that binds the antigen is on the "arms" of the Y, retains its function even when unbound from the Fc fragment

The Fc fragment is the portion that attatches the antigen fragment to the immune cell, the "base" of the Y, retains its function even when unbound from the Fab fragment

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Where does the antigen bind to the antibody?

Hypervariable regions at the two top "tips" of the Y, the antigen binds between the heavy and light chains

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Are the hypervariable regions of antigens on the C- or N-terminus of the protein?

N-terminus of both the heavy and light chains, they run parallel to each other

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What happens when an antibody binds and antigen?

Antibodies are bound at the end of the Fab fragment, can immobilise the antigen by binding to one or two molecules at the same time, preventing them from having their normal motility and activity in the cell. The Fc tail sticks out as a trigger for cellular defense mechanisms

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34

How can we code for 106 antibodies if there are only 20,000 genes?

Antibodies are encoded by the immunoglobulin locus

The immunoglobulin locus contains sets of short gene fragments that are assembled into a complete gene using a special DNA recombination process.

The gene fragments can be combined in many different ways to create a million different antibody genes. 

35

How is the genetic information for the light chain shuffled at the immunoglobulin locus?

The variable (V), joining (J) , and constant (C ) gene fragments reside at the immunoglobulin locus.

When specialising the B cell from the precursor stem cell, special DNA recombination process randomly picks one V region and joins it to one J region to make a mature light chain gene, exising the material in between as a circular loop of DNA.

Transcription of mRNA begins at the last variable segment. The pre-mRNA is spliced so that any remaining upstream joining regions after the first are removed, as well as the intron between the J and C region. The functional mRNA contains only one V, J, and C region and is ready to be translated

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36

How is the genetic information for the light chain shuffled at the immunoglobulin locus?

The heavy chain locus has many V (variable) regions and several J (joining) regions as well as many D (diversity) regions.

When specialising the B cell from the precursor stem cell, special DNA recombination process randomly picks one D region and joins it to one J region. Then another recombination event randomly picks one V region and joins it to the DJ region to make a mature light chain gene, exising the material in between as a circular loop of DNA. The heavy chain is further diversified by the action of terminal deoxyribonucleotidyl transferase, a special DNA polymerase that does not require a template. This enzyme inserts extra nucleotides between VH and D to generate more variants.

Transcription of mRNA begins at the last variable segment. The pre-mRNA is spliced so that any remaining upstream joining regions after the first are removed, as well as the intron between the J and C region. The functional mRNA contains only one V, J, D, and C region and is ready to be translated

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What is an RSS sequence?

Recombination Signal Sequences (RSSs) are found in genomic DNA next to V, D, and J segments. An RSS consists of a heptamer, a spacer, and a nonamer.

The heptamers (7nt, 5’-CACAGTG-3’) and nonamers (9nt, 5’- ACAAAAACC-3’) are brought together and recombination occurs at the ends of the heptamers.

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What size are the spacers in a RSS?

The spacers are either 12 or 23 nucleotides: for any recombination event, one side must have a 12nt spacer and the other a 23nt spacer.

This 12/23 rule prevents V-V recombination, because all V regions at a given locus have the same spacer.

39

What is the difference between when the V(D)J segments are joined compared to when the V(D)JC segments are joined?

The V(D)J segments are joined in DNA through site specific recombination, while the V(D)JC segment is joined to the whole through pre-mRNA splicing, after transcription.

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40

Which regions of the Ig locus code for the pocket that binds antigens?

The V, D, and J regions, which are the more variable regions of the protein

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41

Explain site specific recombination in the DNA of lymphocytes

During V(D)J recombination, the RAG complex (made up of RAG-1 and RAG-2 complexed with HMG1 or HMG2) binds to two recombination signal sequences (RSSs); the complex associates with each other, bringing the strands together, creating a loop which contains all the DNA between the two RSSs. Some of this DNA is then deleted, and the RAG complex then induce a nick precisely at the 5' end of the heptamer. This creates a 3' -OH group which acts as a nucleophile in a transesterification attack on the antiparallel strand, yielding a DNA hairpin (two hairpins, as the RAG complex dimer binds to two strands). In lymphoid cells, recombination can only occur between a 12-RSS and a 23-RSS; this is known as the 12/23 rule.

The four ends of DNA (two hairpinned coding ends and the two signal ends) are held together in a postcleavage complex by the RAG complex. The VDJ recombinase complex can then close up the signal ends into a 'signal joint'. It also opens the hairpins of the coding ends, and this process is thought to be mediated by the RAG complex. Nucleotides are added at the open ends by terminal deoxynucleotidyl transferase (TdT). This occurs until there are complimentary sequences at which point the opposite strands will pair up. Exonucleases then remove the unpaired nucleotides, and ligases fill in the gaps. This creates a junction between each joined segment, containing an unspecified number of nucleotide additions, flanked by a 2-residue palindromic sequence. There is a precise deletion between the heptamers, with release of the excised DNA in circular form 

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combinatorial assembly

Combinatorial assembly uses site specific recombination and protein multimers to make incredible variation out of a small number of genes.  In lymphocytes  ~149 genes can be assembled in 2.4x106 combinations

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Immune tolerance

Process of sensitising the immune system to "self" proteins.

• Early during fetal development, pre-B cells display the particular antibody they will make on their cell surfaces.

• In the fetus, no foreign antigens are present, only ‘self’ molecules.

• If these cells encounter their target antigen, the cells are destroyed. Therefore, no cells will react against self proteins.

• Sometimes, this mechanism fails, leading to autoimmune disease 

44

What ways can further diversity be produced from lymphocytes aside from multimerism and site specific recombination?

Difference mRNA splicing and somatic hypermutation allow even more diversity for antibodies

45

How are antibodies used in Western Blotting?

Proteins are mixed with sodium dodecyl sulfate, which denatures them and gives them a negative charge. They are then passed through polyacrylamide gel electrophoresis, which separates them based on size, as SDS has a charge of -1, and ~1 SDS molecule binds per pair of amino acids. This means heavier compounds will be more negatively charged.

The separated proteins are blotted on nitrocellulose or nylon filters, which are incubated with a primary antibody that recognises the protein of interest.

Another antibody is used to bind the constant region of the first antibody to an enzyme, alkaline phosphatase, which removes phosphate from a colorless dye (XP) that becomes black when dephosphorylated. The bands containing the protein of interest become dark.

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46

Northern Blotting

A general blotting procedure starts with extraction of total RNA from a homogenized tissue sample or from cells.  RNA samples are then separated by gel electrophoresis. Since the gels are fragile and the probes are unable to enter the matrix, the RNA samples, now separated by size, are transferred to a nylon membrane through a capillary or vacuum blotting system.

A nylon membrane with a positive charge is the most effective for use in northern blotting since the negatively charged nucleic acids have a high affinity for them. The transfer buffer used for the blotting usually contains formamide because it lowers the annealing temperature of the probe-RNA interaction, thus eliminating the need for high temperatures, which could cause RNA degradation.[9] Once the RNA has been transferred to the membrane, it is immobilized through covalent linkage to the membrane by UV light or heat. After a probe has been labeled, it is hybridized to the RNA on the membrane.

Probes for northern blotting are composed of nucleic acids with a complementary sequence to all or part of the RNA of interest, they can be DNA, RNA, or oligonucleotides with a minimum of 25 complementary bases to the target sequence. RNA probes (riboprobes) that are transcribed in vitro are able to withstand more rigorous washing steps preventing some of the background noise.

47

What is Northen Blotting used for? Southern? Western?

Northern blotting is used to identify a specific RNA within a mixture of many RNA molecules.

Southern blotting is used to identify a specific DNA within a mixture of many DNA molecules.

Western blotting is used to identify a specific protein within a mixture of many protein molecules.

48

Retrovirus

A class of virii including HIV that use RNA as their genetic material

49

provirus

The host-integrated viral DNA from a retrovirus

50

What ribozyme expresses a provirus?

RNA pol III

51

Normal structural genes found in retrovirii

gag - makes viral capsid proteins

pol - makes viral enzymes: reverse transcriptase, protease

env - makes the outside coat of the virus 

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How is HIV different from other retrovirii?

HIV has all the standard structural genes (gag, pol, env), plus a sophisticated set of regulatory genes. It infects the CD4+ T-cell in the immune system, because the outer proteins of the virus bind to the CD4 molecule and the CCR 5 co-receptor that are present on the surface of this class of T-cells

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gag

gag, short for group-specific antigen, encodes core, nucleocapsid, and matrix proteins

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pol

pol, short for polymerase, encodes the viral enzymes: polymerase, protease, integrase

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env

env , short for envelope, encodes outer glycoproteins: gp120 (binds CD4 and CCR5) and gp41 (involved in fusion)

56

tat

tat, short for transactivator, encodes a positive regulator of HIV transcription

greatly increases steady state levels of HIV mRNAs

•  binds to an RNA element called TAR, present at the 5’ end of all transcripts

•  TAR is a stem/loop structure with a bulge: the Tat protein binds to the bulge while host proteins bind to the loop

•  increases RNA polymerase processivity (RNA polymerase does not fall off DNA template) 

57

rev

rev, short for regulator of viral expression, allows export of unspliced mRNAs from nucleus

58

vif

vif, short for viral infectivity, affects particle infectivity

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vpr

vpr, short for viral protein r, encodes protein for transport of DNA to nucleus, augments virus production, cell cycle arrest

60

vpu

vpu, short for viral protein u, encodes protein that promotes intracellular degradation of CD4 and enhances release of virus from membrane

61

nif

nif, short for negative regulator of infectivity, encodes genes that augments viral replication, downregulates CD4 and MHC class II

62

What are the key events in the HIV life cycle?

1.  fusion with host cell

2.  reverse transcription of viral genome

3.  integration into host genome

4.  transcription

5.  early phase: multiply spliced mRNAs

6.  late phase: export of unspliced mRNAs

7.  gag-pol translational frameshift

8.  cleavage of poly-proteins 

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63

Explain the process of HIV reverse transcription

tRNALEU (packed into the virion) acts as a primer and hybridizes to a complementary part of the virus RNA genome called the primer binding site or PBS

Complementary DNA then binds to the U5 (non-coding region) and R region (a direct repeat found at both ends of the RNA molecule) of the viral RNA

A domain on the reverse transcriptase enzyme called RNAse H degrades the 5’ end of the RNA which removes the U5 and R region

The primer then ‘jumps’ to the 3’ end of the viral genome and the newly synthesised DNA strands hybridizes to the complementary R region on the RNA

The first strand of complementary DNA (cDNA) is extended and the majority of viral RNA is degraded by RNAse H

Once the strand is completed, second strand synthesis is initiated from the viral RNA

There is then another ‘jump’ where the PBS from the second strand hybridizes with the complementary PBS on the first strand

Both strands are extended further and can be incorporated into the hosts genome by the enzyme integrase

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64

Explain the error rate of HIV RTase

HIV RTase is the most error prone DNA polymerase known, with 1 error in 104 bases copied. This is not necessarily bad for the virus!

- helps generate diversity
- helps generate variants that can escape host immune system

Error prone reverse transcriptase generates 106 variants of HIV each day of active infection. 99.9 % of mutations are deleterious, 99% of the remainder are neutral, but this still leaves a few that are beneficial.

If they confer resistance to a drug, the mutant virus can repopulate a person in a few days.

Mathematical modeling suggests that the virus is close to ‘error catastrophe’, where any further increase in mutation rate would be deleterious. 

65

Where is reverse transcription performed?

In the cytoplasm of T-cells

66

What is the difference between tat and a normal transcription factor?

Technically, tat is a transcription factor because it increases the amount of transcription, BUT tat is an RNA-binding protein while traditional transcription factors are DNA-binding proteins!

the tat protein binds to the nascent (newly made) RNA and rides along during transcription, very unusual

•  also seems to increase recruitment of new RNA polymerase to promoter 

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What phase of HIV transcription is tat involved in?

The early phase, tat is a transcription factor that increases recruitment of RNA polymerase to the HIV promoter

68

What is different about the mRNAs exported by HIV-infected cells compared to the mRNAs of a normal cell?

In the late phase of HIV gene expression, mRNAs are exported whole without splicing, which does not occur normally in eukaryotes. The rev protein accomplishes this task.

69

What triggers the late phase of gene expression in HIV?

Once the rev protein accumulates, it triggers the shift to the late phase of gene expression. rev enters the nucleus and binds to an RNA element present in the primary transcript called the RRE (rev-response element). Rev then causes these transcripts to be exported from the nucleus without being spliced. 

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70

Why does HIV begin with multiply spliced mRNAs and then switch to unspliced late mRNAs?

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HIV requires the early protein rev to shuttle unspliced and singly spliced mRNAs out of the nucleus, but multiply spliced mRNAs can leave the nucleus without rev, so the inital mRNAs are small so that intial proteins like rev can be translated in cytoplasm, and return to the nucleus in the later stages to transport the larger transcripts out.

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71

Why does the translational frameshift always occur late in HIV protein synthesis?

The gag/pol region reside on the 9-kb mRNA, which requires rev to shuttle it out of the nucleus.  It cannot be encoded in the early stages.

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72

Why is a translational frameshift required for the synthesis of ______ in HIV?

The virus uses an unusual mechanism to make the pol protein: translational frameshift

gag and pol encoded in same mRNA

• Their ORFs overlap by 200 nucleotides

• They are encoded in different reading frames

• translation starts with gag in one reading frame

• mRNA has a sequence that causes ribosome to slip back one nucleotide (-1 frameshift)

• frameshift only happens 5-10% of the time

• ribosome keeps going in new reading frame

• makes gag-pol fusion protein, later clipped apart 

73

How does HIV force host ribosomes to make a translational frameshift?

To induce frameshifting, the viral mRNA requires a "slippery sequence" (UUUUU) and a small RNA stem/loop structure. These features cause the ribosome to pause, slip backwards one nucleotide, and resume translation in the "-1" reading frame.

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