We are searching data for your request:
Upon completion, a link will appear to access the found materials.
Learning goals associated with Winter_2021_Bis2A_Facciotti_Reading_22
- Differentiate and convert between coding/noncoding, template/non-template strands.
- Define and explain the function and structure of an open reading frame (ORF).
- Create a model for a basic transcriptional unit that includes promoters, transcriptional regulatory sites for transcription factor binding, ribosome binding site, coding region,
stopcodon and transcriptionalterminator.
- Use the model of a transcriptional unit to discuss the roles of each of the structural elements of a transcriptional unit. Identify those that
are transcribed, those that may be translatedand those that serve other roles.
- Use a codon table to “translate” an RNA sequence and
possiblevariants of the sequence into a protein sequence.
- Explain the genetic code means what being
- Diagram the process of translation. The diagram should include reactants (including the
mRNAtemplate and the tRNAs), the products, enzymes, and the sites on the mRNAtemplate required for translationto take place.
- Draw a rough sketch of an
aminoacyl tRNAsynthetase, including its active site and other sites in the enzyme that bind to the reactants.
- Describe the steps in the chemical reaction responsible for
tRNAcharging and lay out its energy story.
- Describe the steps in the chemical reaction responsible for protein synthesis and tell its energy story. Then, compare this story to the one you prepared for DNA synthesis and RNA synthesis.
- Discuss how the primary structure of a protein influences its target destination in the cell for both eukaryotic and bacterial organisms.
The process of translation in biology is the decoding
message into a polypeptide product. Put another way, a message written in the chemical language of nucleotides is "translated" into the chemical language of amino acids. Amino acids
together via covalent bonds (called peptide bonds) between
and carboxyl termini of adjacent amino acids. The decoding and "linking" process
by a ribonucleoprotein complex called the ribosomes and can
chains of amino acids of lengths ranging from tens to over 1,000.
The resulting proteins are so important to the cell that their synthesis consumes more of a cell’s energy than any other metabolic process. Like DNA replication and transcription, translation is a complex molecular process that we can approach using both the Energy Story and Design Challenge rubrics. Describing the overall process, or steps, requires the accounting of the matter and energy before the process and after the process and a description of how that matter
and energy transferred during the process. From a Design Challenge standpoint, we can - even before digging any further into what is or
about translation - try to infer some basic questions that we will need to answer regarding this process.
Let us start by considering the basic problem. We have a strand of RNA (called
) and a bunch of amino
and we need to design a machine that will somehow:
(a) decode the chemical language of nucleotides into the language of amino acids,
(b) join amino acids in a very specific manner,
(c) complete this process with reasonable accuracy, and
(d) do this at a reasonable speed. Reasonable
by natural selection.
As before, we can identify subproblems.
(a) How does our molecular machine determine where and when to work?
(b) How does the molecular machine coordinate decoding and bond formations?
(c) Where does the energy for this process come from and how much?
(d) How does the machine know where to stop?
Other questions and functional problems/challenges will arise as we dig deeper.
The point, as always, is that even knowing no specifics about translation we can use our imaginations, curiosity and common sense to imagine some requirements for the process that we will need to learn more about. Understanding these questions as the context for what follows is key.
A peptide bond links the carboxyl end of one amino acid with the amino end of another, expelling one water molecule. The R1 and R2 designation refer to the side chain of the two amino acids.
Protein Synthesis Machinery
The components that go into the process
Many molecules and macromolecules contribute to the process of translation. While the exact composition of "the players" in the process may vary from species to species - for instance, ribosomes may comprise different numbers of
Reminder: Amino acids
Let us recall that the basic structure of amino acids
The 20 common amino acids.
A ribosome is a complex macromolecule composed of structural and catalytic
, and many distinct polypeptides. As we try thinking about energy accounting in the cell, we note that the ribosomes themselves do not come for "free". Even before
, a cell must invest energy to build each of its ribosomes. In E. coli, there are between 10,000 and 70,000 ribosomes present in each cell.
Ribosomes exist in the cytoplasm in bacteria and archaea and in the cytoplasm and on the rough endoplasmic reticulum in eukaryotes. Mitochondria and chloroplasts also have their own ribosomes in the matrix and stroma, which look more similar to bacterial ribosomes (and have similar drug sensitivities), than the ribosomes just outside their outer membranes in the cytoplasm. Ribosomes dissociate into large and small subunits when they are not synthesizing proteins and reassociate during the initiation of translation. coli, we describe the small subunit as 30S, and the large subunit as 50S. Mammalian ribosomes have a small 40S subunit and a large 60S subunit. The small subunit binds the
template, whereas the large subunit sequentially binds
. Many ribosomes can simultaneously translate an individual
molecule, each ribosome synthesizing protein in the same direction: reading the
from 5' to 3' and synthesizing the polypeptide from the N terminus to the C terminus. The complete
from genes. Depending on the species, 40 to 60 types of
exist in the cytoplasm. Serving as adaptors, specific
bind to sequences on the
template and add the corresponding amino acid to the polypeptide chain. Therefore,
are the molecules that actually “translate” the language of RNA into the language of proteins.
Of the 64 possible
codons—or triplet combinations of A, U, G, and C, three specify the termination of protein synthesis and 61 specify the addition of amino acids to the polypeptide chain. Of these 61, one codon (AUG) also encodes the initiation of translation. Each
anticodon can base pair with one of the
codons and add an amino acid or
translation, according to the genetic code. For instance, if the sequence CUA occurred on
template in the proper reading frame, it would bind a
expressing the complementary sequence, GAU, which would
to the amino acid leucine.
The process of pre-
The Mechanism of Protein Synthesis
Like in transcription, we can divide protein synthesis into three phases: initiation, elongation, and termination. The process of translation is similar in bacteria, archaea and eukaryotes.
In E. coli
, a sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (AGGAGG), interacts with a
molecule. This interaction anchors the 30S ribosomal subunit at the correct location on the
template. Stop for a moment to appreciate the repetition of a mechanism you've encountered before. Here, getting a protein complex to associate - in proper register - with a nucleic acid polymer
by aligning two antiparallel strands of complementary nucleotides with one another. We also saw this in the function of telomerase.
Instead of binding at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 7-
cap at the 5' end of the
. A cap-binding protein (CBP) assists the movement of the ribosome to the 5' cap. Once at the cap, the initiation complex tracks along the
in the 5' to 3' direction, searching for the AUG start codon.
from the first AUG, but this is not always the case. According to Kozak’s rules, the nucleotides around the AUG
whether it is the correct start codon. Kozak’s rules state that the following consensus sequence must appear around the AUG of vertebrate genes: 5'-
-3'. The R (for purine)
a site that can be A or G, but cannot be C or U. Essentially, the closer the sequence is to this consensus, the higher the efficiency of translation.
Possible NB Discussion Point
Compare and contrast the initiation of translation with that of transcription — in what ways are these processes similar and in what ways do they differ?
During translation elongation, the
template provides specificity. As the ribosome moves along the
codon comes into 'view', and
were not present in the elongation complex, the ribosome would bind
nonspecifically. Note again the use of base pairing between two antiparallel strands of complementary nucleotides to bring and keep our molecular machine in register and in this case also to accomplish the job of "translating" between the language of nucleotides and amino acids.
The large ribosomal subunit comprises three compartments:
site binds incoming charged
with their attached specific amino acids), the P site binds charged
carrying amino acids that have formed bonds with the growing polypeptide chain but have not yet dissociated from their corresponding
, and the E site which releases dissociated
so they can
with another free amino acid.
Elongation proceeds with charged
Possible NB Discussion Point
Tetracycline is an antibiotic on the World Health Organization’s List of Essential Medicines. It mitigates infections by blocking the A site on the bacterial ribosome. Another antibiotic, chloramphenicol, blocks peptidyl transfer. Describe the immediate and long-term effects of these two antibiotics. What other strategies can you think of to battle infection specifically at the level of translation?
The Genetic Code
To summarize what we know to this point, the cellular process of transcription generates messenger RNA (
), a mobile molecular copy of one or more genes with an alphabet of A, C, G, and uracil (U). Translation of the
template converts nucleotide-based genetic information into a protein product. Protein sequences
20 commonly occurring amino acids; therefore, we can say that the protein alphabet
20 letters. We define each amino acid by a three-nucleotide sequence called the triplet codon. The relationship between a nucleotide codon and its corresponding amino acid
the genetic code. Given the different numbers of “letters” in the
and protein “alphabets,” means that there are
64 (4 × 4 × 4)
codons; therefore, an amino acid (20 total) must
Three of the 64 codons
protein synthesis and release the polypeptide from the translation machinery. These triplets
stop codons. Another codon, AUG, also has a special function.
specifying the amino acid methionine, it also serves as the start codon to
by the AUG start codon near the 5' end of the
. The genetic code is universal. With a few exceptions, virtually all species use the same genetic code for protein synthesis, which is powerful evidence that all life on Earth shares a common origin.
Redundant, not Ambiguous
The information in the genetic code is redundant. Multiple codons code for the same amino acid. For example, using the chart above, you can find 4 different codons that code for Valine, likewise, there are two codons that code for Leucine, etc. But the code is not ambiguous, meaning, that if
Termination of translation occurs when a stop codon (UAA, UAG, or UGA)
Coupling between Transcription and Translation
As discussed previously, bacteria and archaea need not transport their RNA transcripts between a membrane bound
a protein synthesis Design Challenge we can also raise the question/problem of how proteins get to where they
to go. We know that some proteins
for the plasma membrane, others in eukaryotic cells need to
to various organelles, some proteins, like hormones or nutrient scavenging proteins,
to be secreted by cells while others may need to
to parts of the cytosol to serve structural roles. How does this happen?
Since we have uncovered various mechanisms, the details of this process
in a brief paragraph or two. However, we can mention some key elements common to all mechanisms. First, is the need for a specific "tag" that can provide some molecular information about where the protein of interest
for. This tag usually takes the form of a short string of amino acids - a so-called signal peptide - that can encode information about where the protein should end up. The second required component of the protein sorting machinery must be a system to read and sort the proteins. In bacterial and archaeal systems this usually
proteins that can identify the signal peptide during translation, bind to it, and direct the synthesis of the nascent protein to the plasma membrane. In eukaryotic systems, the sorting is by necessity more complex, and involves a rather elaborate set of mechanisms of signal recognition, protein modification, and trafficking of vesicles between organelles or the membrane. These biochemical steps
in the endoplasmic reticulum and further "refined" in the Golgi apparatus where proteins
and packaged into vesicles bound for various parts of the cell.
. The key for all students it so appreciate the problem and to have a general idea of the high-level requirements that cells have adopted to solve them.
The players in translation include the