Alison Stewart 11/12/06
Prokaryotic Cells, Eukaryotic cells and HIV:
Structures, Transcription and Transport
Section Handout Discussion Week #7
Compare and contrast the organization of eukaryotic, prokaryotic and HIV genomes:
size
Proteins per gene
Gene organization
Coding sequence
Human
3.3 x 109 bp
1
‘random’
Discontinuous
Introns/exons
E. coli
4.6 X 106
1 or more
‘random’ + operons
Continuous
(no introns)
HIV
9000 bp
1 or more
?
Both
This is not to memorize, but more to clearly display the differences. You should know
that eukaryotic genes have exons/introns, but generally prokaryotes do not.
How the HIV genome packs so much information into a tiny genome?
1) overlapping genes e.g. gag and pol
2) genes separated so splicing must occur (we will get to splicing, it basically
means cutting and pasting a gene or RNA together from two or more different
segments) e.g rev and tat
Also HIV does not need to code for everything since it is not self sufficient.
HIV genes to know:
gag
Erin
env
Erin
pol
Rob/David
tat
Rob
rev
Rob
Envelope protein, helps budding
Encodes gp120 and gp41
Encodes reverse transcriptase, integrase, and protease
Enhances transcription
Nuclear export
Cellular/Viral particle Structure:
Prokaryotic Cell
Outside:
plasma membrane – phospholipid bilayer
cell wall - (remember transpeptidase, it helps build the cell wall), keeps shape of cell
periplasmic space - small space in between membranes or in this case between cell
wall and plasma membrane
Inside:
NO compartments - (there are LOTS of prokaryotes so this is a generalization)
Genetic material is DNA - organized into nucleoid NO MEMBRANE, but has associated
nucleoid proteins not naked DNA
Eukaryotic Cell
Outside:
plasma membrane – phospholipid bilayer
Inside:
Compartments- called organelles, they are also surrounded by membranes
Genetic material is DNA – contained in nucleus, bound up by proteins called histones
Other important organelles:
(These are the important ones, but not key to memorize unless mentioned in class)
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Mitochondria - energy generators of the cell - they harness energy from food
to make ATP - the chemical that fuels cell activities; surrounded by a double
membrane (therefore have periplasmic space!)
Endoplasmic Reticulum, (ER) - a maze of interconnected spaces surrounded
by a membrane, the site of synthesis of proteins destined for membranes. The
ER is contiguous with the nuclear membrane, but this does NOT mean that the
the proteins, lipids, etc. in the two membranes are the same.
Golgi apparatus - a stack of flattened disks of membrane that receives proteins
from the ER, modifies them and directs them to other organelles, the plasma
membrane or to the exterior of the cell (carried in secretory vesicles).
Lysosomes - sites of degradation of macromolecules (a sort of trash can)
Peroxisomes - a contained environment for reactions involving hydrogen
peroxide, a highly reactive molecule.
Transport in and out of organelles
Nucleus – has pores so that small molecules and ions can freely cross and diffuse from
cytosol, but NOT large proteins or nucleic acids (they must be transported)
Other organelles, e.g. ER and mitochondria – no pores, must have special transport for
ions, small molecules, and proteins
Transport of proteins occurs in lipid vesicles within the secretory pathway (more on this
later)
Virus
Outside:
structural proteins – for HIV gp41 and gp120 are important, they are glycoproteins
plasma membrane – phospholipid bilayer
Inside:
Capsid- made of protein, it protects proteins and genome inside until release is
appropriate for infection
Genetic material – for HIV it is RNA, in general viruses can have RNA or DNA
genomes, also associated with proteins so it is not “naked” when injected into a human
cell
Intracellular Trafficking – pertinent to eukaryotes and HIV
viral particle maturation
Key components of the secretory pathway:
ER
Golgi
Plasma membrane
Lysosomes
Endosomes – come from outside the cell and bud into the cell with outside stuff
Targeting:
Targeting occurs by signal sequences, these signals are recognized by transport
receptors. There is at least one transport receptor for each compartment.
Target sequences can be contiguous or not contiguous if the amino acids are close in
the folded up tertiary structure.
The SNARE pathway is an example of how the target sequence causes proteins to be
transported to other compartments. See the image.
1) Cargo binds to a cargo receptor that associates with a vesicle-SNARE
2) A vesicle covered with v-SNAREs finds a compartment with the
complementary target-SNAREs.
3) The v-SNARE binds the t-SNARE which causes a folding into a bundle of 4
helices that brings the membranes together for fusion.
This is analogous, but not the same as the hairpin intermediate formed in HIV entry and
fusion.
Targeting to ER
From the ER proteins go to lysosomes, the golgi and the plasma membrane (remember
endosomes come from the outside). As soon as a signal sequence is synthesized the
nascent polypeptide can go into the ER, even if the protein is not finished being made.
Water soluble and transmembrane proteins can be made in the ER. However, once
proteins go in the ER they typically do NOT go back out into the cytosol. They can be
put in the plasma membrane or another compartment or outside of the cell.
HIV takes advantage of this targeting to get its membrane proteins made and
glycosylated. The HIV protein gp160 is a membrane protein that is the precursor of
gp120 and gp41. It has an N-terminal sequence that directs it to the ER.
Post-translational modifications:
Post-translational refers to after a gene (DNA) is transcribed into RNA then translated
into a protein made of amino acids, then any subsequent changes are post-translational.
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Signal sequences targeting to the ER are cleaved in the ER by signal peptidase.
Glycosylation, the addition of oligosaccharides is performed.
In addition, the ER has proteins that assist in folding properly and the formation of the
correct disulfide bonds.
The N-terminal signal of gp160 is cleaved, then at least some of its 30 potential N-linked
glycosylation sites (which amino acid does this mean!?!) are glycosylated by enzymes
and 10 disulfide bonds are formed. Without these changes the protein does not
function properly and can aggregate.
The Golgi apparatus
Vesicles from the ER are targeted to the Golgi by proteins that coat the vesicles. Exactly
which proteins when is not key, but here is an image showing that process.
In the Golgi there is further glycosylation - addition, modification and removal of sugars
to the overall oligosaccharide occurs.
The cis-Golgi is near the ER and the trans Golgi is across the rest of the Golgi from the
ER. As proteins in the Golgi move from the cis to the trans Golgi this is when processing
occurs since there are different proteins in the different compartments. The contents of
the compartments are overlapping so this is a general trend rather than a strict rule.
At the trans-Golgi proteins leave and get sorted to different locations, such as to the
plasma membrane.
In the Golgi gp160 glycosylation is modified, then in the trans-Golgi it is cleaved into
gp120 and gp41, which are targeted to the plasma membrane and travel via secretory
vesicles.
To finish the HIV story:
Viral assembly and exit from the cell Gag is a cytoplasmic protein, once it is modified by the addition of a fatty acid tail it
goes to the inner leaflet of the membrane. There it attracts the viral genome and is
presumed to help deform the plasma membrane and the viral particle buds off with
gp41 and gp120 on the outside.
What is missing from this picture? Draw it in.
Transcription:
Prokaryotic transcription:
Initiation:
Promoters – are often at specific locations, -10 and -35 upstream from the
transcriptional start site (position +1).
Transcriptional start sites are thus dictated by the promoter site.
Transcription factors – specific sigma factors (s) bind to the -10 and -35 site and recruit
RNA polymerase to that location.
RNA polymerase – catalyzes the polymerization of new RNA strands.
Termination: At specific sequences (termination sequences), the newly synthesized
RNA will fold onto itself due to self-complementarity. This will create a hairpin structure
that will help the newly synthesized RNA ‘push’ off RNA polymerase from the RNA/DNA
hybrid. This is not always how it happens, but the example for you to remember.
Eukaryotic transcription:
Promoters – You can refer to the entire regulatory region (core promoter that has
binding site for RNA polymerase complex and also DNA that has binding site(s) for
transcription factors) as a promoter.
Transcription factors – function like the Sigma factor, but instead of one protein being
used, many proteins bind and assemble together to perform the same function. They
recruit and activate RNA Pol to a specific location (gene).
RNA Polymerase – performs the same function as bacterial RNA polymerase. mRNA
transcription uses RNA Pol II.