Transcript The Process of Transcription-2

Storage and use of genetic information
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• The genetic code
– Three bases (in a row) specify an amino acid
• Transcription
– The synthesis of a mRNA, complementary to one of
the DNA strands, containing the genetic code
• Translation
– Proteins and rRNAs in the ribosome along with
tRNAs translate the genetic code into proteins.
• Post-translational modification
– Proteins are altered after synthesis
The Genetic Code
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• Four bases taken how many at a time? Need to
code for 20 different amino acids.
– Each base = 1 amino acid: only 4
– Every 2 bases = 1 a.a.: 16 combinations, 4 short.
– Every 3 bases: 64 combinations, enough.
• Every 3 bases of RNA nucleotides: codon
– Each codon is complementary to 3 bases in one
strand of DNA
– Each codon (except for T →U switch) is the same
as 3 bases in the other DNA strand.
More about the Genetic Code
• The code is
– Unambiguous: each codon specifies 1 amino acid
– Degenerate: a particular amino acid can be coded for by
several different codons.
– Ordered: similar codons specify the same amino acid.
– Commaless, spaceless, and non-overlapping : each 3
bases is read one after the other.
– Punctuated: certain codons specify “start” and “stop”.
– Universal: by viruses, both prokaryotic domains, and
eukaryotes (except for some protozoa, mitochondria).
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The Genetic Code-2
http://www.biology.arizona.edu/molecular_bio/problem_sets/nucleic_acids/graphics/gencode.gif
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Wobble
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• Crick’s Wobble Hypothesis
• The code is “ordered”
– The first 2 positions are more important
– When lining up with the anticodon of the tRNA, the third
position doesn’t bind as tightly, thus a looser match is
possible.
– Because of this flexibility, a cell doesn’t need 61
different tRNAs (one for each codon).
• Bacteria have 30-40 different tRNAs
• Plants, animals have up to 50.
Colinearity
• Archibald Garrod (1908) determined that genes must
code for enzymes by studying metabolic diseases
(black urine). But what relationship?
• Beadle and Tatum (1941): one gene one enzyme,
there is a one to one correspondence between DNA
and protein
– Mutation in DNA changes nucleotide sequence,
changes amino acid sequence in protein.
– Not completely true in eukaryotes because of
introns
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The code is colinear with protein sequence
• Bacteriophage MS2
– An RNA virus with 3 genes, including a viral coat
protein.
– Amino acid sequence of coat protein determined,
1970
– Sequence of coat protein gene determined, 1972
– Correspondence between codons and amino acids
exactly as predicted.
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Ribosome structure
Large and small
subunits.
Eukaryotic:
60S & 40S = 80S
Prokaryotic:
50S & 30S = 70 S
Large subunit: 2 -3 rRNAs and many proteins
Small subunit: 1 rRNA and many proteins
http://www.emc.maricopa.edu/faculty/farabee/BIOBK/ribosome.gif
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Size in Svedbergs
• S is a unit based on ultracentrifugation
– What affects how fast particles move toward the
bottom of the tube?
– Mass, density, and shape (which affects friction)
• Example: supercoiled DNA moves faster than
relaxed DNA.
– Because of these factors, S units are not additive
• 50S + 30S prokaryotic subunits make a 70S
ribosome.
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Components of ribosomes
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• Multiple copies exist of ribosomal genes
– Def. of “gene” extended to DNA that codes for rRNA
– Multiple copies means moderately repetitive DNA
– Cells can crank out lots or rRNA (and r-proteins)
• Transcription results in RNA requiring
processing
– Pre-RNA cut into rRNAs (and tRNAs)
• rRNA NOT just structural: are ribozymes, carry
out the actual protein synthesis.
About tRNAs-1
• Coded for by tRNA genes
– Post-transcriptional modification
– tRNAs have some bases changed
• tRNAs interact with rRNA during protein
synthesis
• tRNAs must have amino acid attached
– Enzymes: aminoacyl tRNA synthetases
• 20 different enzymes, one for each aa.
– Enzymes recognize shape of tRNA
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About tRNAs-2
• tRNA structure must be highly conserved
– Must be folded so that both the synthetases
recognize it, and it fits properly on the ribosome.
– Errors in translation result in bad proteins;
mutations in tRNA genes are selected against.
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tRNA
3D structure:
The familiar
loops of the
2D structure
are labeled.
Decoder end:
Complementary to
codon.
hto-b.usc.edu/~cbmp/2001/ tRNA/trna%20s1.jpg
3’ end:
Attaches to
amino acid.
Translation
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mRNA: provides message to be translated.
Ribosomes: functional workbench for synthesis.
tRNA: bring aa to ribosome, decode mRNA.
Aminoacyl tRNA synthetases: enzymes that attach
amino acids to tRNAs.
• Protein factors: help move process along: initiation,
elongation, and termination.
• Process is similar, but different between prokaryotes
and eukaryotes.
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Initiation and Termination of protein
synthesis
formyl
• AUG is always the first codon (initiator codon)
– Establishes an “open reading frame” (ORF)
– Ribosome begins synthesis with a methionine
• In bacteria, it is N-formylmethionine (fMet)
• After synthesis , either formyl group is removed or
entire fMet is removed (Met in eukaryotes)
• Three codons serve as termination codons:
– UGA, UAG, UAA; any one can be a stop signal
– Do NOT code for an amino acid
– Cause translation to end; protein is completed
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Translation-1
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• Initiation
– Small subunit, mRNA, met-tRNA, IFs, GTP
– mRNA: sequence for binding to ribosome needed
• prokaryotes: Shine-Delgarno
• Eukaryotes: Cap and Kozak sequence
– (GCC)RCCATGG where R is a purine
– First tRNA is fMet-tRNA in prokaryotes
– IFs are protein Initiation Factors
– GTP needed for energy
– When all have come together, Large subunit added
Translation-2
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• Ribosome has 3 sites
– AA site where tRNA-aa first sits in
– P site where tRNA with growing peptide sits
– E or Exit, site transiently occupied by used tRNA
• Elongation, with help of EFs and GTP
– tRNA with new aa sits in A site
– Stays in A site if anticodon on tRNA is
complementary to codon on mRNA.
– tRNA in P site transfers growing chain to new aa
• Catalyzed by rRNA
• Ribosome moves relative to mRNA and tRNAs
– tRNAs now in new sites, new codon lined up
Ribosome schematic
http://staff.jccc.net/pdecell/proteinsynthesis/translation/elongation12.gif
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Translation-3
• Termination
– When stop codon is in A site, no tRNA binds
– GTP-dependent release factor (protein) removes
polypeptide from tRNA in P site. All done.
– Ribosomal subunits typically dissociate.
• Do a Google Search for translation animation
– Many hits. Note presence, absence of E site
– Note shape of ribosomes
– Note whether role of rRNA in catalysis is shown
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Nonsense mutations and suppressors
• A mutation may change
a normal codon to a stop
codon; protein synthesis
ends prematurely.
(nonsense mutation)
A second mutation can cure
the original: a “suppressor”.
If the gene for a tRNA is
mutated in the anticodon
so that the stop codon is
now read by the tRNA.
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Polysomes and Polycistronic mRNA
• In eukaryotes, when mRNA enters the
cytoplasm, many ribosomes attach to begin
translation. A mRNA w/ many ribosomes
attached = polysome.
• In eukaryotes, the mRNA for a single gene is
processed and translated; in prokaryotes,
mRNA can be polycistronic, meaning several
genes are on the same mRNA and are
translated together
– With no nucleus, translation can begin in
prokaryotes before transcription is over.
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Polysomes
Multiple ribosomes attach to the
mRNA and begin translating.
Strings of ribosomes can be seen
attached to the mRNA.
http://opbs.okstate.edu/~petracek/
Chapter%2027%20Figures/Fig%2
027-29b-bottom.GIF
www.cu.lu/labext/rcms/
cppe/traducti/tpoly.html
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Eukaryotic, prokaryotic differences
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mRNA lifetime is different
Cap, tail, introns in eukaryotes
Shine-Delgarno vs. Kozak sequence & cap
Size of ribosomes: 70S vs. 80S
fMet-tRNA vs. Met-tRNA
Eukaryotic: attachment of ribosomes to ER
– Polypeptides extruded through tunnel in large
subunit, directly into ER
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Proteins vs. polypeptide
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• Common usage:
– Polypeptide is a string of amino acids
• Doesn’t imply function
• Doesn’t imply 3D shape
– Protein implies functionality and 3D shape
• Thus a “protein” can have a quaternary structure
and be made of several different polypeptides.
Review of protein structure
String of amino acids, covalently attached by peptide
bonds; directional (N terminus, C terminus).
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Primary structure
The particular amino acids and
the order that they are in.
20 different amino acids connected by peptide bonds;
100-1000 amino acids in a peptide chain.
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Secondary structure
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Amino acid chain twists in space, held in
place by hydrogen bonds. Forms alpha
helix or beta pleated sheet.
Tertiary structure
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3-D folding of the protein chain
in space; the shape is
determined from the primary
structure and from the
secondary structure (which
itself depends on the primary
structure.
The primary structure depends
on the info encoded in the DNA
Protein structure slides from
Quaternary structure
Many proteins that act on
DNA or RNA have a
quaternary structure.
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Individual polypeptide
chains aggregate to
form a single functional
unit. Individual
polypeptides (protein
subunits) may be
switched out to give the
multipart protein a
different function or
specificity.
Domains
Different exons actually
code for parts of proteins
that fold into discrete
areas.
May be involved in
evolution of proteins and
their functions.
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