Summary of Essential Cell Biology by Alberts et al. including chapters 7,8,10-14. Also including extra's from slides and wikipedia + exams from other years and practice questions. Made for Biochemie @ VU for SBI, MNW, mBio, S and FAR.
Chapter 7: From DNA to Protein ................................................................................... 2
Eucaryotic Transcription ......................................................................................................... 2
Non‐coding sequences .............................................................................................................. 3
Translation ................................................................................................................................... 4
Chapter 8 Control of Gene Expression ........................................................................ 6
How Transcriptional Switches Work ................................................................................... 6
The molecular mechanisms that create specialized cell types ................................... 7
Post‐Transcriptional Controls ............................................................................................... 7
Chapter 10 Analyzing Genes and Genomes ............................................................... 8
Manipulating and Analyzing DNA Molecules..................................................................... 8
DNA Cloning .................................................................................................................................. 9
Deciphering and Exploiting Genetic Information ......................................................... 10
Chapter 11 Membrane Structure............................................................................... 12
Chapter 12 Membrane Transport ............................................................................. 15
Principles of Membrane Transport ................................................................................... 15
Transporters and Their Functions .................................................................................... 16
Ion Channels and the Membrane Potential..................................................................... 17
Ion Channels and Signaling in Nerve Cells ...................................................................... 17
Chapter 13 How Cells Obtain Energy from Food .................................................. 18
The Breakdown and Utilization of Sugars and Fats ..................................................... 18
Regulation of Metabolism ..................................................................................................... 21
Chapter 14 Energy Generation in Mitochondria and Chloroplasts................ 22
Mitochondria and Oxidative Phosphorylation .............................................................. 22
Chloroplasts and Photosynthesis ....................................................................................... 25
The Origins of Chloroplasts and Mitochondria ............................................................. 25
Literature .......................................................................................................................... 25
,Chapter 7: From DNA to Protein
How Cells read the Genome
Genes direct the synthesis of proteins. This occurs through the intermediate (m)RNA.
Transcription = the process of copying a DNA‐sequence (gene) into RNA
DNA RNA
Deoxyribonucleotides: A‐T and C‐G Ribonucleotides: A‐U and C‐G
Double stranded helix Single stranded (free‐foldable)
Information storage Transcription, catalysis, structural
Max. 250M nucleotides Max 2‐5k nucleotides
Transcription is carried out by RNA polymerases, which catalyze the formation of
phosphodiester bonds that link the nucleotides together and form the sugar‐
phosphate backbone of the RNA chain. The energy needed for these additions is
derived from triphosphates covalently bound to the incoming ribonucleotides. RNA
polymerase further moves from 5’ to 3’ on the template strain (DNA).
5’= is phosphate end which is attached to the fifth carbon of the (deoxy‐)ribose
3’= is the (deoxy‐)ribose end where nucleophile groups can attack the third carbon
atom
Genes code for different types of RNA:
mRNA: Messenger RNA is translated to proteins by RNA polymerase II
tRNA: Transfer RNA forms the adaptor needed in translation by ribosomes
rRNA: Ribosomal RNA forms the core of ribosomes
miRNA: Micro RNA regulates gene expression by binding to mRNA’s 5 UTR
RNA‐polymerase starts transcriptions at promoters on the DNA. It stops at a
terminator where it releases both DNA and newly made RNA.
For bacterial polymerase (just 1 type), a subunit called the sigma factor recognizes
the promoter sequence of DNA to start transcription. Eukaryotic RNA polymerase
has no sigma factor but requires a greater set of general transcription factors to start
transcribing. Also unlike prokaryotes, eukaryotic DNA has more space between
genes, allowing more complex forms of transcriptional regulation. Finally bacterial
DNA is not packed into nucleosomes and chromatin.
Eucaryotic Transcription
As in DNA replication, DNA is read from 3' → 5' during transcription. Meanwhile, the
complementary RNA is created from the 5'UTR → 3'UTR direction (Un‐Translated
Region). This means its 5' end is created first in base pairing. Although DNA is
arranged as two antiparallel strands in a double helix, only one of the two DNA
strands, called the template strand, is used for transcription. This is because RNA is
only single‐stranded, as opposed to double‐stranded DNA. The other DNA strand is
called the coding (lagging) strand, because its sequence is the same as the newly
,created RNA transcript (except for the substitution of uracil for thymine). The use of
only the 3'UTR → 5'UTR strand eliminates the need for the Okazaki fragments seen
in DNA replication.1
At the TATA‐box (25 nucleotides upstream of the first nucleotide to be transcripted
and present on the coding/lagging strand) the first general transcription factor
(GTF) TFIID assembles and distorts the DNA to function as a landmark for other
factors to assemble there. The total of all factors including RNA polymerase II is
called the transcription initiation complex. The RNA polymerase II is released when
its tail is phosphorylated. When transcription is finished a phosphatase will strip off
the phosphates so it can attach to a new promoter.
In bacterial transcription, translation starts immediately as DNA and RNA are free in
cytosol and ribosomes are not separated by membrane of the nucleus.
In eukaryotic transcription the RNA first needs to travel out of the nucleus. Secondly
the RNA must undergo RNA processing steps (capping and polyadenylation).
Enzymes on the tail of RNA polymerase perform these processes while transcribing.
For mRNA the two processing steps are:
RNA‐capping: a guanine‐nucleotide with a methyl‐group is attached after about 25
nucleotides of transcription to the 5’ end of the RNA
Polyadenylation: RNA is cut off and gets an addition of a few hundred andenine‐
nucleotides.
These functional groups help stabilize, recognize and transport the mRNA.
Non‐coding sequences
Bacteria have continuous genes, meaning the full gene is the message itself. In
eukaryotic DNA, coding stretches (exons) are interrupted by non‐coding stretches
(introns). These genes consist in majority of introns.
To produce mRNA the full gene in transcribed including introns. These introns are
removed by RNA splicing. Elaborate splicing machines, spliceosome (RNA), mostly
cut out the introns by recognizing particular sequences at the end of an intron. The
removed introns are called lariats. The spliceosome consists of small nuclear RNA
(snRNA) and additional proteins. The complex is called snRNP and forms the heart of
spliceosome.
The intron‐exon structure allows alternative splicing, meaning that different proteins
can be produced from a single gene. Furthermore it allows genetic recombination of
exons to be more likely. Exons usually code for certain protein domains, thus introns
allow new configurations of proteins to be made (evolutionary advantage).
Splicing is possible due to the nucleophilic second carbon of the ribose sugar
contained in the nucleotide.
After splicing and capping an mRNA is functional and can move towards the cytosol.
,This is a selective process and the mRNAs will need the proper protein attachments
(e.g. the cap and adenyl‐tail) to exit through the nuclear pores. All debris is being
degraded as a result of eukaryotic transcription.
Its 3’ UTR sequence and the cell it is produced in determine the amount of time an
mRNA exists in the cytosol. Bacterial mRNA degrades rapidly in minutes as
eukaryotic mRNA can last between 30 minutes and 10 hours.
Translation
From RNA to protein; the genetic code
Nucleotide triplets/codons code for amino acids; the building blocks of proteins.
There are 43=64 combinations. However there are combinations reserved for stop
codons and different combinations of nucleotides code most amino acids.
The adaptor molecule tRNA (+‐80nt) can bind to the codons and bind with a specific
amino acid. The tRNA folds to a 3D structure as it will undergo base pairing with
itself. The 4 unfolded parts are functional areas. One of them is the anti‐codon.
The anti‐codon is the tRNA region that binds to an mRNA‐codon. Bare in mind that
the anti‐codon from 5’‐3’ has to be flipped and reversed to know its bound amino
acid. There are more than one tRNAs that bind with the same codon and there are
tRNAs that bind to more than one codon (not ambiguous but redundant). Most
tRNAs only recognize the first two nucleotides of a codon. A mismatch on the third
position, a wobble, can be tolerated as all 4 combinations code for the same amino
acid. Hence the last nucleotide of the codon differs in codons that code for the same
amino acid.
As tRNA is somewhat unspecific to codon binding, the aminoacyl‐tRNA synthetase
enzyme covalently binds one specific amino acid only to tRNAs that code for that
amino acid. This enzyme recognizes the last codon at the 3’ end.
The high‐energy bond created between the tRNA and the amino acid by the use of
ATP will in a later stage be used for the linking of the protein through a peptide
bond.
Ribosomes read from 5’ to 3’ the mRNA and link the amino acids of tRNAs together.
They are built up of rRNA and ribosomal proteins. The proteins stabilize the catalyst
rRNA core, which functions as a ribozyme: an RNA molecule functioning as an
enzyme. This functionality is an argument for the current paradigm that RNA
predates DNA. Experiments also show that the ribozyme can function without its
protein subunits.
As a whole it consist of two parts. The smaller unit that matches tRNA to the codons
on the mRNA and the larger unit that catalyzes the formation of peptide bonds to
covalently link the amino acids present on the tRNAs.
Entering tRNA is bound at the A site. It moves on to the P‐site of the ribosome where
the amino acid will be transferred and exits after it moved to the E‐site. This process
,is repeated until a stop codon is reached. mRNA is bound to the small ribosomal
subunit.
For the ribosome, the reading frame (1/3) is determined by start codon AUG coding
for methionine. This results in each protein having a methionine residue at the N
terminus. Most of the time this amino acid is removed by specific proteases.
The initiator tRNA is a special tRNA carrying the methionine amino acid. It binds
together with the small ribosomal subunit and translation initiation factors. When a
AUG codon is found, the TIFs dissociate and the large ribosomal unit will bind to start
translation.
Bacterial mRNA needs no cap for ribosomes to start searching for the start codon.
However they have ribosome binding sites. These sites are necessary as bacterial
mRNAs are often polycistronic, meaning one mRNA codes for multiple proteins.
The stop codon codes for a protein called the release factor (RF), which doesn’t
attach an amino acid to polypeptide chain. With the RF bound the peptidyl
transferase in the ribosome, it will now catalyze the addition of water, thus finishing
the protein at the C terminus and releasing it into the cytosol.
Molecular chaperones meet the released protein at the ribosomes to help it
properly fold into the desired 3D structure.
To amplify protein production, multiple ribosomes may translate the protein at the
same time as close as 80nt from each other. mRNAs with multiple ribosomes
attached are called polyribosomes/polysomes.
For the production of antibiotics fungi are being used as they are also eukaryotic like
humans. They create toxins that inhibit protein synthesis in bacteria. Because that
process is slightly different than synthesis in eukaryotes, it is non‐toxic to humans
and fungi.
To regulate the amount of proteins, the lifetime of these molecules has to be
controlled. Proteins are being recycled and degraded by proteases and
proteasomes. Proteasomes recognize ubiquitin (a small protein), which is covalently
attached to proteins that are marked for destruction.
,Chapter 8 Control of Gene Expression
All cells of an individual have the same genome, however not all genes are expressed
in each sell. This results in differentiation.
Different cells express different genes and thus different proteins (epigenome).
However a lot of housekeeping proteins are the same for each cell. Each different
cell type produces its specialized enzymes, which are unique to that cell.
External signals, like hormones, can induce changes in gene expression. This reaction
can be different among different cells and may not even induce a reaction.
Gene expression can be controlled at different steps in the pathway from DNA to
RNA to protein. Transcriptional control is the most effective and most important.
Fig. 1: Control of gene expression on multiple levels
How Transcriptional Switches Work
Proteins binding to regulatory DNA sequences control transcription. This can either
be by inhibiting (repressor) the initiation site or by requirement of different
transcription factors (activator), which all have to be present to start transcription.
Nearly all genes in prokaryotes and eukaryotes have regulatory DNA sequences (10‐
10.000 np) upstream of the initiation site.
To have any effect, these sequences must bind transcription regulators. The
simplest bacterium codes for several hundred transcription regulators each of them
binding to a specific sequence of DNA, thereby regulating specific genes. These
proteins are quite unique, but often consist of certain common structures that bind
to DNA in the major groove. These subunits are called DNA‐binding motifs, e.g.
homeodomain, zinc finger and the leucine zipper.
Transcription switches allow cells to respond to changes in the environment. In
bacteria, multiple genes are often coded into one mRNA (polyprotein). Such a set of
genes is called an operon. For example the tryptophan repressor is transcription
regulator for the operon of tryptophan enzymes. It binds to a DNA sequence called
the operator and prevents binding of RNA‐polymerase to the promoter. However it
,only binds when it’s under allosteric control of a number of tryptophan molecules
(negative feedback). Also activators are under allosteric control. Some repressors in
bacteria can also be activators at other parts of the DNA.
Unregulated genes are called constitutive genes and operate constantly at low level.
Eukaryotic transcription regulators control gene expression from a distance. These
are called enhancers and can influence the rate of transcription. They can either be
up or downstream at a distance of several thousand base pairs (bp). They can either
distort or activate the transcription initiation complex. The DNA needs to be looped
to reach this site and is often helped by mediator proteins that connect them
together.
Finally transcription may also be under epigenetic control. Many transcription
regulators can also attract histone (de‐)acetylases, which can modify the histone tail,
making the DNA more or less accessible.
The molecular mechanisms that create specialized cell types
When cells have differentiated, they pass on their gene expression to next
generations as well, this called cell memory. In contrast some the simplest changes
in expression are only transient.
Many eukaryotic genes are under combinatorial control, meaning different proteins
work together to determine the gene expression of a single gene.
Also one ligand can control different transcription regulator, for example the
hormone cortisol can control all the genes that code for tyrosine aminotransferase.
This functioning is also how cells differentiate.
There are three ways to inherit gene expressions from a mother cell:
‐ Through positive feedback loop a transcription regulator can regulate its own
gene, which codes it.
‐ Through faithful propagation of a condensed chromatin structure
‐ Through DNA‐methylation. Enzymes copy the pattern of methylation
(epigenome) during division. For vertebrates the cytosine base is methylated.
Post‐Transcriptional Controls
Riboswitches: Are part of some genes mRNA, mostly in bacteria, and can act during
transcription as a cut off switch when it is bound to metabolite. Mostly the
metabolite for which the enzyme of the gene is coded produces. Riboswitches are
again proof that RNA was able to regulate protein production before DNA‐world.
RNA‐splicing: see previous chapter
miRNA: Are small stretches of RNA that basepair with mRNAs, blocking their
translation or otherwise marking them for cleanup in the RNA‐Induced‐Silencing‐
Complex: RISC. They bind mostly to the untranslated parts of mRNA and can regulate
the next mRNA after the current one is removed.
RNAi: Some proteins that process miRNAs also serve as a cell defense mechanism.
The nuclease Dicer cuts the foreign double stranded RNA into pieces called siRNAs.
These stretches are incorporated into the RISC and then basepair with single strands
of foreign RNA and will be removed. RNAis might be used for determining gene
, functions and might even be used in treating diseases as it gives the potential to
switch genes off.
Chapter 10 Analyzing Genes and Genomes
Manipulating and Analyzing DNA Molecules
Before it was hard to understand the functioning of genes, because they don’t exist
as a separate entity, unlike proteins. The solution was given by the discovery of
restriction nucleases, which cut the DNA at certain points of specific short
nucleotide sequences (palindromes). They can produce a reproducible set of
fragments from DNA.
Fig.2: Restriction enzymes cut at specific palindrome sequences
Different bacteria contain different restriction nucleases, each cutting at a different
sequence for any DNA. Because they are short stretches, by chance a number of
sites will be present for fragmentation. Typical enzymes may be chosen for the
production of certain desired DNA‐fragments. Plasmids are also designed to contain
only unique palindrome sequences to insert genes at only one specific place.
Gel electrophoresis separates DNA fragments (negatively charged) of different sizes
on a polyacrylamide gel. Small fragments move to the positive electrode better than
larger pieces. The DNA bands on the agarose slab are invisible, so after separation
they need to be labeled or stained. Staining can be done by exposing DNA to a dye
that fluoresces under UV‐light. Even better detection is possible with radiolabeling P‐
32 and incorporates it into the DNA and finally making an autoradiograph by X‐ray
photography.
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