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Winter_2021_Bis2A_Facciotti_Reading_22 - Biology

Winter_2021_Bis2A_Facciotti_Reading_22 - Biology



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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 andtranscriptionalterminator.
  • Use the model of a transcriptional unit to discuss the roles of each of the structural elements of a transcriptional unit. Identify those thatare transcribed, those that maybe translatedand those that serve other roles.
  • Use a codon table to “translate” an RNA sequence andpossiblevariants of the sequence into a protein sequence.
  • Explain the genetic code means what beingdegenerate.
  • Diagram the process of translation. The diagram should include reactants (including themRNAtemplate and thetRNAs), the products, enzymes, and the sites on themRNAtemplate required fortranslationto take place.
  • Draw a rough sketch of anaminoacyltRNAsynthetase, including its active site and other sites in the enzyme that bind to the reactants.
  • Describe the steps in the chemical reaction responsible fortRNAcharging 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.

Protein Synthesis

Introduction

The process of translation in biology is the decoding

anmRNA

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

are linearly strung

together via covalent bonds (called peptide bonds) between

amino

and carboxyl termini of adjacent amino acids. The decoding and "linking" process

is catalyzed

by a ribonucleoprotein complex called the ribosomes and can

result in

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

is transformed

and energy transferred during the process. From a Design Challenge standpoint, we can - even before digging any further into what is or

is not understood

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

mRNA

) and a bunch of amino

acids

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

is defined

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.
Attribution:Marc T. Facciotti (original work).

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 rRNAs (ribosomal RNAs) and polypeptides depending on the organism - the general functions of the protein synthesis machinery are comparable from bacteria to human cells. We focus on these similarities. At a minimum, translation requiresanmRNAtemplate, amino acids, ribosomes, tRNAs, an energy source, and various additional accessory enzymes and small molecules.

Reminder: Amino acids

Let us recall that the basic structure of amino acidsconsists ofa backbone composed of an amino group, a central carbon (called theα-carbon), and a carboxyl group. Attached to theα-carbon is a variable group that helps determine some chemical properties and reactivity of the amino acid.

A generic amino acid.
Attribution:Marc T. Facciotti (own work)

The 20 common amino acids.
Attribution:Marc T. Facciotti (own work)

Ribosomes

A ribosome is a complex macromolecule composed of structural and catalytic

rRNAs

, 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

anmRNAis translated

, 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

mRNA

template, whereas the large subunit sequentially binds

tRNAs

. Many ribosomes can simultaneously translate an individual

mRNA

molecule, each ribosome synthesizing protein in the same direction: reading the

mRNA

from 5' to 3' and synthesizing the polypeptide from the N terminus to the C terminus. The complete

mRNA

/poly-ribosome structure

is called

a polysome.

The protein synthesis machinery includes the large and small subunits of the ribosome,mRNA, andtRNA.
Source: http://bio1151.nicerweb.com/Locked/m.../ribosome.html

TRNAs

tRNAs are structural RNA molecules that

were transcribed

from genes. Depending on the species, 40 to 60 types of

tRNAs

exist in the cytoplasm. Serving as adaptors, specific

tRNAs

bind to sequences on the

mRNA

template and add the corresponding amino acid to the polypeptide chain. Therefore,

tRNAs

are the molecules that actually “translate” the language of RNA into the language of proteins.

Of the 64 possible

mRNA

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

tRNA

anticodon can base pair with one of the

mRNA

codons and add an amino acid or

terminate

translation, according to the genetic code. For instance, if the sequence CUA occurred on

anmRNA

template in the proper reading frame, it would bind a

tRNA

expressing the complementary sequence, GAU, which would

be linked

to the amino acid leucine.

The folded secondary structure of atRNA.The anticodon loop and amino acid acceptor stem are indicated.
Source: http://mol-biol4masters.masters.grkr...ansfer_RNA.htm

Aminoacyl tRNASynthetases

The process of pre-tRNAsynthesis by RNA polymerase III only creates the RNA portion of the adaptor molecule. The corresponding amino acid mustbe addedlater, once thetRNAis processedand exported to the cytoplasm. Through the process oftRNA“charging,” eachtRNAmoleculeis linkedto its correct amino acid by a group of enzymes called aminoacyl tRNAsynthetases. At least one type ofaminoacyl tRNAsynthetase exists for each of the 20 amino acids;theexact number ofaminoacyl tRNAsynthetases varies by species. These enzymes first bind and hydrolyze ATP tocatalyzea high-energy bond between an amino acid and adenosine monophosphate (AMP);theyexpel a pyrophosphate molecule in this reaction. The activated amino acidis then transferredto thetRNA, andAMP is released.

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.

Translation Initiation

Generally, proteinsynthesis begins with the formation of an initiation complex. The small ribosomal subunit will bind to themRNAat the ribosomal binding site. Soon after, the methionine-tRNAwill bind to the AUG start codon (through complementary binding with its anticodon). This complexis then joinedby large ribosomal subunit. This initiation complex then recruits the secondtRNAandthus translation begins.

Translation begins when atRNAanticodon recognizes a codon on themRNA. The large ribosomal subunit joins the small subunit, anda second tRNA is recruited. As themRNAmoves relative to the ribosome,the polypeptide chain is formed. Entry of a release factor intothe Asiteterminatestranslation and the components dissociate.

BacterialvsEukaryoticinitiation

In E. coli

mRNA

, a sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (AGGAGG), interacts with a

rRNA

molecule. This interaction anchors the 30S ribosomal subunit at the correct location on the

mRNA

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

is accomplished

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-

methylguanosine

cap at the 5' end of the

mRNA

. 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

mRNA

in the 5' to 3' direction, searching for the AUG start codon.

Many eukaryotic mRNAs are translated

from the first AUG, but this is not always the case. According to Kozak’s rules, the nucleotides around the AUG

indicate

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'-

gccRccAUGG

-3'. The R (for purine)

indicates

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?


Translation Elongation

During translation elongation, the

mRNA

template provides specificity. As the ribosome moves along the

mRNA

, each

mRNA

codon comes into 'view', and

specific binding with the corresponding charged tRNA anticodon is ensured

. If

mRNA

were not present in the elongation complex, the ribosome would bind

tRNAs

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:

the A

site binds incoming charged

tRNAs

(

tRNAs

with their attached specific amino acids), the P site binds charged

tRNAs

carrying amino acids that have formed bonds with the growing polypeptide chain but have not yet dissociated from their corresponding

tRNA

, and the E site which releases dissociated

tRNAs

so they can

be recharged

with another free amino acid.

Elongation proceeds with chargedtRNAsenteringthe Asite and then shifting to the P site followed by the E site with each single-codon “step” of the ribosome.Ribosomal steps are inducedby conformational changes that advance the ribosome by three bases in the 3' direction. The energy for each step of the ribosomeis donatedby an elongation factor that hydrolyzes GTP. Peptide bonds form between the amino group of the amino acid attached tothe A-sitetRNAand the carboxyl group of the amino acid attached to the P-sitetRNA. The formation of each peptide bondis catalyzedby peptidyltransferase, an RNA-based enzyme thatis integratedinto the 50S ribosomal subunit.The energy for each peptide bond formation is derivedfrom GTP hydrolysis, whichis catalyzedby a separate elongation factor. The amino acid bound to the P-sitetRNAis also linkedto the growing polypeptide chain. As the ribosome steps across themRNA, the former P-sitetRNAenters the E site, detaches from the amino acid, andis expelled. The ribosome moves along themRNA, one codon at a time,catalyzingeach process that occurs in the three sites. With each step, a chargedtRNAenters the complex, the polypeptide becomes one amino acid longer, and an unchargedtRNAdeparts. Amazingly, this process occurs rapidly in the cell, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200-amino acid polypeptide couldbe translatedin just 10 seconds.


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 (

mRNA

), a mobile molecular copy of one or more genes with an alphabet of A, C, G, and uracil (U). Translation of the

mRNA

template converts nucleotide-based genetic information into a protein product. Protein sequences

consist of

20 commonly occurring amino acids; therefore, we can say that the protein alphabet

consists of

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

is called

the genetic code. Given the different numbers of “letters” in the

mRNA

and protein “alphabets,” means that there are

a total of

64 (4 × 4 × 4)

possible

codons; therefore, an amino acid (20 total) must

be encoded

for by

more than

one codon.

Three of the 64 codons

terminate

protein synthesis and release the polypeptide from the translation machinery. These triplets

are called

stop codons. Another codon, AUG, also has a special function.

In addition to

specifying the amino acid methionine, it also serves as the start codon to

initiate

translation.

The reading frame for translation is set

by the AUG start codon near the 5' end of the

mRNA

. 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.

This figure shows the genetic code for translating each nucleotide triplet, or codon, inmRNAinto an amino acid or a termination signal in a nascent protein. (credit: modification of work by NIH)
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 ifyou were givenacodonyou would know definitively which amino acid it is coding for, a codon will only code for a specific amino acid. For example, GUU will always code for Valine, and AUG will always code for Methionine. This is important,you will be askedto translateanmRNAinto a protein using a codon chart like the one shown above.

Translation Termination

Termination of translation occurs when a stop codon (UAA, UAG, or UGA)is encountered. When the ribosome encounters the stopcodonnotRNAentersthe Asite. Instead, a protein known as a release factor binds to the complex. This interaction destabilizes the translation machinery, causing the release of the polypeptide and the dissociation of the ribosome subunits from themRNA. After many ribosomes have completed translation, themRNAis degradedsothe nucleotides can be reusedin another transcription reaction.

Coupling between Transcription and Translation

As discussed previously, bacteria and archaea need not transport their RNA transcripts between a membrane boundnucleousand the cytoplasm. The RNA polymerase is therefore transcribing RNA directly into the cytoplasm. Here ribosomes can bind to the RNA and begin the process of translation, sometimes while transcription is still occurring. The coupling of these two processes, and evenmRNAdegradation,is facilitatednot only because transcription and translation happen in the same compartment but also because both of the processes happen in the same direction - synthesis of the RNA transcript happens in the 5' to 3' direction and translation reads the transcript in the 5' to 3' direction. This "coupling" of transcription with translation occurs in both bacteria and archaea and is, in fact, essential for proper gene expression sometimes.

Multiple polymerases can transcribe a single bacterial gene whilenumerousribosomes concurrently translate themRNAtranscripts into polypeptides. In this way, a specific protein can rapidly reach a high concentration in the bacterial cell.

Protein Sorting

In

the context of

a protein synthesis Design Challenge we can also raise the question/problem of how proteins get to where they

are supposed

to go. We know that some proteins

are destined

for the plasma membrane, others in eukaryotic cells need to

be directed

to various organelles, some proteins, like hormones or nutrient scavenging proteins,

are intended

to be secreted by cells while others may need to

be directed

to parts of the cytosol to serve structural roles. How does this happen?

Since we have uncovered various mechanisms, the details of this process

are not easily summarized

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

is destined

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

consists of

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

are initiated

in the endoplasmic reticulum and further "refined" in the Golgi apparatus where proteins

are modified

and packaged into vesicles bound for various parts of the cell.

Some specific mechanisms may be discussed by your instructor in class

. 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.

Post-translationalProtein Modification

Aftertranslationindividual amino acids maybe chemically modified. These modifications add chemical variation and new properties thatare rootedin the chemistries of the functional groups that are being added. Common modifications include phosphate groups,methyl,acetate, andamidegroups. Some proteins, typically targeted to membranes, will belipidated-a lipid will be added. Other proteins willbe glycosylated- a sugar willbe added. Another common post-translational modification iscleavageor linking of parts of the protein itself. Signal-peptides maybe cleaved, parts maybe excisedfrom the middle of the protein, ornew covalent linkages may be madebetween cysteine or other amino acid side chains. Enzymes willcatalyzenearly all modifications and all change modifications the functional behavior of the protein.

Section Summary

mRNAis used to synthesize proteins by the process of translation. The genetic code is the correspondence between the three-nucleotidemRNAcodon and an amino acid. The genetic code is “translated” by thetRNAmolecules, which associate a specific codon with a specific amino acid. The genetic code isdegeneratebecause 64 triplet codons inmRNAspecify only 20 amino acids and three stop codons. This means thatmore thanone codon corresponds to an amino acid. Almost every species on the planet uses the same genetic code.


The players in translation include themRNAtemplate, ribosomes,tRNAs, and various enzymatic factors. The small ribosomal subunit binds to themRNAtemplate. Translation begins at the starting AUG on themRNA. The formation of bonds occurs between sequential amino acids specified by themRNAtemplate according to the genetic code. The ribosome accepts chargedtRNAs, and as it steps along themRNA, it catalyzes bonding between the new amino acid and the end of the growing polypeptide.The entiremRNAis translatedin three-nucleotide “steps” of the ribosome. When a stop codonis encountered, a release factor binds and dissociates the components and frees the new protein.