lecture 24
protein synthesis stage 3 - elongation sequence what happens after translocation?
the fMet-tRNA leaves the E site. the fMet-aa1 dipeptide occupies the P site. ready for another round of elongation using the same materials
aminoacyl-transfer-RNA synthetases what does the synthetase use to prevent the attachment of incorrect amino acids?
the functional groups and shape of its cognate amino acid
protein synthesis stage 3 - peptidyl transfer what does the mechanism of peptidyl transfer from aminoacyl-tRNA to the growing peptide chain involve?
an amino group from the aminoacyl-tRNA in the A site performing a nucleophilic attack on the carbonyl carbon of the aminoacyl-tRNA (peptide) in the P site
protein synthesis stage 3 - peptide bond formation what happens when a tRNA is attached to the growing peptide chain in the P site?
an aminoacyl-tRNA binds to the A site
3rd step of protein synthesis what is elongation?
an energy requiring process that occurs on and is catalyzed by ribosomes
aminoacyl-transfer-RNA synthetases what does this create?
an ester linkage between the carboxyl group of an amino acid and the 2'- or 3'-hydroxyl group of the adenosine unit of a CCA sequence at the 3' end of the tRNA
aminoacyl-transfer-RNA synthetases how many specific aminoacyl-transfer-RNA synthetase and tRNA for each amino acid?
at least one each
tRNA how much is there for each of the 20 amino acids?
at least one kind of tRNA for each of the 20 amino acids
how many tRNAs are there for each amino acid?
at least one. many amino acids have more than one bc every organism has at least 30-50 tRNA and there is only 20 amino acids
translation in eukaryotes where does initiation occur?
at the AUG closest to the 5" end of the mRNA. the 40S finds this site by binding to the 5' cap and then scanning the RNA until AUG is reached
tRNA how is the amino acid recognized in translation?
bc of the complementation between an anticodon triplet on the tRNA and a codon triplet on the mRNA template
protein synthesis stage 3 - elongation why is elongation very accurate?
bc of the specific base pairing between the anticodon triplet on the aminoacyl-tRNA's codon units on the mRNA
protein synthesis stage 2 - assembly of initiation complex what happens in the first part of the initiation complex?
binds to the shine-dalgarno sequence near the 5' end of the mRNA and the fmet-tRNA becomes oriented on the first AUG codon. IF 3 has completed it tasks and goes away
protein synthesis stage 3 - elongation sequence how is the amino acid delivered to the A site?
by elongation factor TU (EF-Tu) in a GTP-dependent reaction
protein synthesis stage 3 - elongation how is the transfer of amino acid residues from the tRNA's to the growing peptide chain catalyzed?
by ribozyme (catalytic RNA) activity in the core region of the ribosome
protein synthesis stage 3 - elongation sequence what is the peptide bond formation catalyzed by?
by ribozyme activity of the 23S RNA of the large ribosomal subunit. only catalyzed by RNA, no protein is at this site
how has the 50S ribosome structure been characterized?
by x-ray crystallography
tRNAs function
carriers of activated amino acids. also contains anticodon regions that recognize distinct codons in the mRNA
what is tRNAs role?
carries amino acids in an activated form to the ribosome for peptide-bond formation
what does the mutation cause?
causes a dramatic change in the solubility of hemoglobin
substitution what is a missence mutation?
change in one amino acid. very little effect on the function of protein
mutations what does frameshift cause?
changes in the amino acid sequence of the protein product bc the reading frame is shifted. also results in the formation of a shortened protein molecule
tendency to hybridize within the same chain
characteristic in my RNAs
what are stop codons?
codons that code for termination of translation. they don't code for amino acids. when translation machinery reaches one of these three codons, translation ceases
rRNA what does rRNA do?
constitutes the major component of ribosomes
tRNA DHU loop
contains modified base dyhydrouridine
what is something unique tRNAs sometime contain?
distinct and rare bases. including methylated derivatives of A, U, G, C, psedouracil, and dihydrouracil
protein synthesis stage 3 - elongation sequence what is translocation catalyzed by?
elongation factor G (EF-G) in another GTP-dependent reaction
3rd step of protein synthesis
elongation happens. requires an mRNA template and various charged tRNAs
protein synthesis stage 2 - shine-dalgarno in initiation what would happen if initiation happened at a different AUG?
elongation would occur with the wrong reading frame, which would produce a meaningless truncated protein
mRNA what is a distinct mRNA produced for?
for each gene in higher eukaryotes
what are mutations?
genetic alterations in DNA structure
tRNA anticodon loop
has a nucleotide triplet (anticodon) which recognizes a specific amino acid codon in the mRNA. contributes to the amino acid specificity of a given tRNA
protein synthesis stage 4 - polyribosomes how is the length of the protein products made?
if the ribosome is towards the 5' end of the mRNA the products would be shorter. if it happens further down the mRNA, the products will be longer. it is longest at the 3' end
50S what is the structure of the ribosome?
in the center core there are 23S rRNA and the 5S rRNA. there is a site of peptide bond synthesis that lies within a central cavity that only contains the 2 rRNAs and no proteins
tRNA T-PseudoU-C loop
involved in binding to the ribosome. has the rare pseudouridine. this sequence also has a T that is normally not found in RNA
mRNA what can it code for?
it can code for individual genes in bacteria but it usually codes for a cluster of related genes
what is mRNA's role in translation?
it carries nucleotide sequence information that can be translated into a sequence of amino acids in the final protein product
what does the mutation do?
it changes a CTT sequence in the DNA to a CAT. that alters the encoded amino acid from glutamate to valine
protein synthesis stage 1 where does the amino acid attach to the tRNA?
it covalently links to the 3' hydroxyl of a ribose by an ester bond
tRNA how big is tRNA?
it is the smallest RNA. length is only 75 bases
protein synthesis stage 3 - elongation sequence what does the energetics mean?
it requires the hydrolysis of 4 phosphoanhydride bonds to forge one peptide bond during protein synthesis
how does the mutation cause sickle cell?
it results in an amino acid change at position 6 in the beta chain of hemoglobin
protein synthesis stage 2 - shine-dalgarno in initiation why is SD sequence important?
it results in initiation at the nearest AUG
translation which direction is mRNA synthesized in? what about proteins?
mRNA: 5' to 3; protein: amino to carboxyl direction
translation in eukaryotes what is the initiating amino acid group?
methionine
16S what constitutes the mismatched pairs?
mostly GU pairs that are a purine-pyrimidine pair
protein synthesis stage 2 - initiation what is the first amino acid at the N-terminal end of proteins in prokaryotes?
n-formylmethionine
16S is complementation between the stems 100%?
no
normal RBC vs sickle cell
normal: biconcave disc sickle: long rod-like structure that distorts the shape of cell
protein synthesis stage 2 - initiation where does initiation happen?
occurs downstream from a purine rich Shine-Dalgarno sequence on the mRNA
mutations what is frameshift mutation?
occurs when a base is inserted or deleted from a sequence of DNA.
translation where does translation take place?
on ribosomes, which are ribonucleoprotein particles consisting of large and small subunits
how big are tRNAs?
pretty small. has 70-80 nucleotides
speculation about protein synthesis?
primitive organisms may have used only RNA to catalyze protein synthesis and ribosomes were added later
50S what is peptide bond formation catalyzed by?
rRNA without the direct involvement of any catalytic protein
what does aminoacyl-transfer-RNA synthetases do?
reads the genetic code
protein synthesis stage 4 - termination what recognizes the stop codons?
releasing or termination proteins
frameshift
results in very negative changes in the character and function of the protein product
what is the pseudouridine structure like?
same basic structure as uridine, point of attachment between the base and the ribose is changed. instead of a C-N bond there is a C-C bond. can still hydrogen bond but specificity has changed
properties of the genetic code the code is read form the 5' to 3' end of the mRNA
same direction as DNA and RNA synthesis
properties of the genetic code the code is degenerate
several different codons specify the same amino acid. there are 64 ways to code for 20 amino acids
what are tRNAs specifically?
small adaptors that link the triplet codon in mRNA to an amino acid in protein
protein synthesis stage 3 what is the wobble hypothesis?
some aminoacyl-tRNAs recognize more than one codon because pairing of the 3rd base of a codon is less crucial than that of the other 2
protein synthesis stage 3 - wobble hypothesis how many bases can tRNA read?
some can read up to three triplets. true when the 5' base on the anti-codon is the non-standard inosine molecule
aminoacyl-transfer-RNA synthetases how can incorrect amino acids be removed?
some synthetases have separate active sites where they can be removed
protein synthesis stage 3 what is the starting and end point?
starts at the amino terminal end ends at the carboxyl terminal end
what is sickle cell anemia caused by?
substitution mutation
protein synthesis stage 2 - initiation what is the first AUG codon downstream from the Shine-Dalgarno sequence recognized by?
tRNA carrying N-formylmethionine
protein synthesis stage 3 - elongation sequence what are the energetics of peptide bond formation?
takes 2 GTP's converting to GDP and Pi to drive the elongation phase. It takes one ATP converting to AMP and 2 Pi's to charge each amino acid onto its tRNA
protein synthesis stage 3 - elongation sequence where does elongation take place and what is it catalyzed by?
takes place on the ribosome and is catalyzed by a ribozyme (catalytic RNA) enzyme
4th step of protein synthesis
termination. occurs when the translation machinery encounters a stop codon in the mRNA
protein synthesis stage 2 - shine-dalgarno in initiation what does the SD sequence ensure?
that protein synthesis will proceed from the correct start codon and in the right reading frame
1st stage of protein synthesis what is the important feature?
that the correct amino acid is charged onto the appropriate tRNA
50S what does the x-ray data suggest?
that there are various proteins around the periphery of the ribosome
1st stage of protein synthesis
the 20 different amino acid substrates undergo activation by the formation of a covalent aminoacyl-tRNA derivatives
tRNA what is the site of amino acid attachment?
the 3' hydroxyl group on the end of the stem
protein synthesis stage 3 - elongation sequence what recognizes the AUG codon of the mRNA in the first fMet?
the 3'-UAC-5' anticodon sequence of the fMet-tRNA
protein synthesis stage 3 - wobble hypothesis what does the wobble hypothesis suggest?
the 5' base on the anti-codon triplet, which binds to the 3' base on the mRMA triplet, is not as spatially confined as the other two bases. so it could exhibit non-standard base pairing
protein synthesis stage 2 - assembly of initiation complex what happens in the second part of the initiation complex?
the 50S ribosomal subunit is bound and the IF 1 and IF 2 are released. hydrolysis of GTP to GDP and Pi also occurs. complete 70S ribosome is formed and fMet-tRNA is positioned on the first AUG codon
translation in eukaryotes what is the ribosome like?
the 80S ribosome has a 40S small subunit and a 60S large subunit
protein synthesis stage 2 - initiation what is the only AUG in a gene that is read as N-formylmethionine?
the AUG codon at the 5' end of the coding sequence
substitution what is a silent mutation?
the DNA and RNA are changed but the protein is not altered
protein synthesis stage 4 - polyribosomes what is a polyribosome/polysome?
the activity of multiple ribosomes carrying out protein synthesis on a single mRNA
protein synthesis stage 3 - elongation what does the process of elongation involve?
the addition of sequential amino acid residues to the growing peptide chain
protein synthesis stage 3 - wobble hypothesis example of wobble with 3 alanine codons
the alanyl-tRNA (carries the anticodon 3'-CGI, inosine on first base), recognizes the codons 5'-GCU, 5'-GCC, and 5'-GCA
aminoacyl-transfer-RNA synthetases what does this activate and link?
the amino acids to a specific tRNA
aminoacyl-transfer-RNA synthetases what does this recognize?
the anticodon, the acceptor stem, and sometimes other parts of its tRNA substrate
protein synthesis stage 3 - peptide bond formation how are the codons of mRNA recognized (form base pairs with)?
the anticodons of the aminoacyl-tRNAs
mutations what does substitution affect?
the codon for a single amino acid. it will not change codons for any other amino acids bc the reading frame is not shifted
properties of the genetic code the genetic code is comma-less
the codons of 3 bases are read continuously with no spacer bases in between them. the first 3 bases are read, then the next 3, etc
protein synthesis stage 2 - synthesis of formylmethionyl-tRNA what is the formyl group donor in the 2nd reaction?
the coenzyme N^10-formyl tetrahydrofolate (derivative of folic acid)
protein synthesis stage 1 amino acid activation
the covalent attachment of an amino acid to a tRNA is an energy requiring process that is coupled to the hydrolysis of ATP to AMP and inorganic pyrophosphate. An aminoacyl-AMP serves as an activated intermediate in the process
protein synthesis stage 3 - elongation sequence what happens during translocation?
the deacylated tRNA moves to the E site and the peptidyl-tRNA moves from the A site into the P site
protein synthesis stage 3 - peptide bond formation how does the tRNA move?
the deacylated tRNA moves to the E site and then leaves the ribosome. the peptidyl-tRNA moves from the A site to the P site
what is the structure of dihydrouridine like?
the double bond found in uridine ring is reduced. the reduced ring of dyhydrouridine is bent because it is no longer aromatic. dyhydrouridine is less capable of hydrogen binding with other bases
what is the synthesis ribosome like in eukaryotes?
80S ribosome with a mass of 3500 kd
protein synthesis stage 3 - peptide bond formation what are the three sites ribosomes have for tRNA binding?
A (aminoacyl) site, the P (peptidyl) site, and E (exit) site
What is the Shine-Dalgarno sequence?
A short conserved nucleotide sequence upstream of the AUG start codon that serves to align the mRNA on the bacterial small ribosomal subunit
what is the codon for methionine?
AUG
translation what is the start signal on prokaryotic mRNA?
AUG (or GUG). it's preceded by a purine-rich sequence that can base-pair with 16S rRNA
what are ribonucleoproteins?
two-thirds RNA and one-third protein
what is methionine's role in proteins?
usually the first amino acid incorporated into a protein
protein synthesis stage 2 - initiation what else does initiation involve?
various proteins and the 50S ribosomal subunit and requires GTP hydrolysis
how accurate is translation?
very accurate. the incorporation of an incorrect amino acid is less than 10^-4
mutations what is substitution (point) mutation?
when one base is substituted fir another in the DNA sequence.
protein synthesis stage 4 - termination when is protein synthesis terminated?
when one of the stop codons (UAG, UAA, UGA) is encountered in the mRNA sequence
protein synthesis stage 3 - peptide bond formation how is a peptide bond formed?
when the amino group of the aminoacyl-tRNA attacks the ester carbonyl group of the peptidyl-tRNA
rRNA what are the 3 kinds of rRNA in e.coli?
23S, 16S, 5S. one molecule of each is present in each ribosome
how many stem loop structures are in tRNA?
3
how many stages are in protein synthesis?
4
protein synthesis stage 3 what direction are the codons read?
5' to 3'
what do the ribosomes look like?
50S: baseball glove 30S: legless ghost 70S: madonna and child
protein synthesis stage 3 - wobble hypothesis who made up the wobble hypothesis?
Francis Crick in 1966
what is a stem-loop structure?
The RNA structure where there is a helical stem and a loop at the top as the result of RNA folding back on itself on one strand
what are the stop codons?
UAA, UGA, UAG
what is the synthesis ribosome like in prokaryotes?
a 70S ribosome with a mass of 2700 kd
rRNA what is rRNAs role in protein synthesis?
a catalytic and structural role
what is a polysome?
a cluster of ribosomes held together by a strand of mRNA that each ribosome is translating.
protein synthesis stage 2 - initiation what does the shine-dalgarno sequence bind to?
a complementary region on the 16S component of the 30S ribosome
protein synthesis stage 2 - initiation what are the other AUG codons recognized by?
a different tRNA (tRNAm) that carries and delivers normal methionine residue
properties of the genetic code the genetic code is non-overlapping?
a given stretch of mRNA has only one reading frame and only codes for one protein. The mRNA is translated three bases at the time from a fixed point to yield a single defined protein product
mRNA what do mRNAs consist of?
a heterogenous class of molecules whose size reflects the size of the proteins they encode. in e.coli they are 1.2 kilobases
protein synthesis stage 2 - assembly of initiation complex how is the 30S complex formed (prokaryotes)?
a mRNA, fMet-tRNAf, GTP, and a 30S ribosomal subunit all come together with the assistance of 3 initiation factor proteins (IF 1, IF 2, IF 3)
translation in eukaryotes what does regulation of translation provide?
a means for regulating gene expression
protein synthesis stage 3 - elongation sequence what does the amino group of the aminoacyl-tRNA at the A site attacking the ester carbonyl group of the fMet-tRNA at the P site form?
a peptide bond
protein synthesis stage 3 - peptidyl transfer what is the result?
a peptide bond is formed and the link between the peptide in the P site and its tRNA is severed
protein synthesis stage 2 - synthesis of formylmethionyl-tRNA what forms methionyl-tRNA
a specific methionyl-tRNA synthetase
protein synthesis stage 2 - synthesis of formylmethionyl-tRNA what happens in the 2nd reaction?
a trans formylase enzyme adds a one carbon formyl group to the a-amino group of the methionine residue
properties of the genetic code a sequence of 3 bases in mRNA carries the code for one amino acid
a triplet of nucleotides (codon) in mRNA is translated into one amino acid in the protein
properties of the genetic code the code is universal
all organisms use essentially the same code. the same codons specify identical amino acids in the most ancient single celled organism and in humans
what is translation mediated by?
the interplay of more than a hundred macromolecules. includes mRNA, rRNAs, tRNAs, aminoacyl-tRNA synthetases, and protein factors
protein synthesis stage 4 - termination what happens after hydrolysis?
the link to the tRNA is severed and the protein goes away. the ribosomal complex dissociates from the mRNA
what is translation (protein synthesis)?
the mechanism by which the nucleotide sequence information in mRNA is used to produce the amino acid sequence of a protein
protein synthesis stage 3 - elongation sequence what happens when an fMet -tRNA is attached to the P site on the ribosome?
the next aminoacyl-tRNA binds to the A site
what is anemia?
the reduced capacity to transport oxygen in the blood
protein synthesis stage 3 - elongation sequence what must happen after peptide bond formation for the next cycle to begin?
the tRNA's must be translocated
protein synthesis stage 3 - peptide bond formation what happens when the peptide-bond is formed?
the tRNAs and the mRNA have to be translocated for the next cycle to begin
what is mRNAs rolde?
the template for protein synthesis
what does protein synthesis involve?
the translation of nucleotide sequences into amino acid sequences
2nd stage of protein synthesis
there is a specific and accurate mechanism for the initiation of protein synthesis. this occurs at the first AUG next to a purine rich Shine-Delgarno sequence in the mRNA
translation what is translation and transcription like in prokaryotes?
they are closely coupled. several ribosomes can simultaneously translate the same mRNA, forming a polysome
what happens with the distorted cells?
they burst and the hemoglobin is degraded. causes anemia
protein synthesis stage 1 what do the 2 classes of synthetases do?
they each recognize 10 amino acids. the recognize opposite faces of tRNA molecules
protein synthesis stage 4 - termination what do the termination factors do?
they hydrolyze the bond between the proteins and the tRNA
protein synthesis stage 4 - polyribosomes can several ribosomes translate the same mRNA at the same time?
yes