15: Genes and Proteins

Question 1

What is the Central Dogma?

Answer 1

It states that genes specify the sequence of mRNAs, which in turn specify the sequence of proteins.

Question 2

What is a codon?

Answer 2

Three consecutive nucleotides in mRNA that specify the insertion of an amino acid or the release of a polypeptide chain during translation.

Question 3

What does it mean to be colinear?

Answer 3

In terms of RNA and protein, three “units” of RNA (nucleotides) specify one “unit” of protein (amino acid) in a consecutive fashion.

Question 4

What is degeneracy?

Answer 4

(of the genetic code) describes that a given amino acid can be encoded by more than one nucleotide triplet; the code is degenerate, but not ambiguous.

Question 5

What is a nonsense codon?

Answer 5

One of the three mRNA codons that specifies termination of translation.

Question 6

What is a reading frame?

Answer 6

A sequence of triplet codons in mRNA that specify a particular protein; a ribosome shift of one or two nucleotides in either direction completely abolishes synthesis of that protein.

Question 7

What is the genetic code?

Answer 7

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 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, it can be said that the protein alphabet consists of 20 letters. Each amino acid is defined by a three-nucleotide sequence called the triplet codon.

Question 8

What are amino acids?

Answer 8

There 20 amino acids used in protein synthesis, each composed of an amino group (NH₃⁺), a carboxyl group (COO^–), and a side chain (R). The side chain may be nonpolar/polar, charged/uncharged, small/large, acidic/basic. Variation in amino acid sequence gives rise to enormous variation in protein structure and function.

Question 9

How were codons experimentally demonstrated?

Answer 9

Francis Crick and Sydney Brenner used the chemical mutagen proflavin to insert one, two, or three nucleotides into the gene of a virus. When one or two nucleotides were inserted, protein synthesis was completely abolished. When three nucleotides were inserted, the protein was synthesized and functional. This demonstrated that three nucleotides specify each amino acid.

Question 10

Which types of codons have special functions?

Answer 10

In addition to instructing the addition of a specific amino acid to a polypeptide chain, three of the 64 codons terminate protein synthesis and release the polypeptide from the translation machinery. These triplets are called nonsense codons, or 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.

Question 11

How conserved is the genetic code?

Answer 11

The genetic code is universal. With a few exceptions, virtually all species use the same genetic code for protein synthesis. Conservation of codons means that a purified mRNA encoding the globin protein in horses could be transferred to a tulip cell, and the tulip would synthesize horse globin. That there is only one genetic code is powerful evidence that all of life on Earth shares a common origin, especially considering that there are about 10⁸⁴ possible combinations of 20 amino acids and 64 triplet codons.

Question 12

Why is the genetic code degenerate?

Answer 12

Degeneracy is believed to be a cellular mechanism to reduce the negative impact of random mutations. Codons that specify the same amino acid typically only differ by one nucleotide. In addition, amino acids with chemically similar side chains are encoded by similar codons. This nuance of the genetic code ensures that a single-nucleotide substitution mutation might either specify the same amino acid but have no effect or specify a similar amino acid, preventing the protein from being rendered completely nonfunctional.

Question 13

What is consensus?

Answer 13

A DNA sequence that is used by many species to perform the same or similar functions.

Question 14

What is a core enzyme?

Answer 14

A prokaryotic RNA polymerase consisting of α, α, β, and β’ but missing σ; this complex performs elongation.

Question 15

What is downstream?

Answer 15

Nucleotides following the initiation site in the direction of mRNA transcription; in general, sequences that are toward the 3’ end relative to a site on the mRNA.

Question 16

What is a hairpin?

Answer 16

The structure of RNA when it folds back on itself and forms intramolecular hydrogen bonds between complementary nucleotides

Question 17

What is a holoenzyme?

Answer 17

A prokaryotic RNA polymerase consisting of α, α, β, β’, and σ; this complex is responsible for transcription initiation.

Question 18

What is an initiation site?

Answer 18

A nucleotide from which mRNA synthesis proceeds in the 5’ to 3’ direction, denoted with a “+1”.

Question 19

What is a non-template strand?

Answer 19

A strand of DNA that is not used to transcribe mRNA; this strand is identical to the mRNA except that T nucleotides in the DNA are replaced by U nucleotides in the mRNA.

Question 20

What is a plasmid?

Answer 20

Extrachromosomal, covalently closed, circular DNA molecule that may only contain one or a few genes; common in prokaryotes.

Question 21

What is a promoter?

Answer 21

A DNA sequence to which RNA polymerase and associated factors bind and initiate transcription.

Question 22

What is Rho-dependent termination?

Answer 22

In prokaryotes, termination of transcription by an interaction between RNA polymerase and the rho protein at a run of G nucleotides on the DNA template.

Question 23

What is Rho-independent termination?

Answer 23

Sequence-dependent termination of prokaryotic mRNA synthesis caused by hairpin formation in the mRNA that stalls the polymerase.

Question 24

What is a TATA box?

Answer 24

A conserved promoter sequence in both eukaryotes and prokaryotes that helps to establish the initiation site for transcription.

Question 25

What is a template strand?

Answer 25

A strand of DNA that specifies the complementary mRNA molecule.

Question 26

What is a transcription bubble?

Answer 26

A region of locally unwound DNA that allows for transcription of mRNA.

Question 27

What is upstream?

Answer 27

Nucleotides preceding the initiation site; in general, sequences toward the 5’ end relative to a site on the mRNA.

Question 28

How do prokaryotic genomes differ from eukaryotes?

Answer 28

A bacterial chromosome is a covalently closed circle that, unlike eukaryotic chromosomes, is not organized around histone proteins. The central region of the cell in which prokaryotic DNA resides is called the nucleoid. In addition, prokaryotes often have abundant plasmids, which are shorter circular DNA molecules that may only contain one or a few genes. Plasmids can be transferred independently of the bacterial chromosome during cell division and often carry traits such as antibiotic resistance.

Question 29

How are downstream and upstream denoted?

Answer 29

Downstream nucleotides are denoted with positive (“+”) numbers and upstream nucleotides are denoted with negative (“–”) numbers.

Question 30

How is the efficiency of transcription in prokaryotes increased?

Answer 30

Prokaryotes do not have membrane-enclosed nuclei. Therefore, the processes of transcription, translation, and mRNA degradation can all occur simultaneously. The intracellular level of a bacterial protein can quickly be amplified by multiple transcription and translation events occurring concurrently on the same DNA template. Prokaryotic transcription often covers more than one gene and produces polycistronic mRNAs that specify more than one protein.

Question 31

How is the prokaryotic RNA polymerase organized?

Answer 31

Prokaryotes use the same RNA polymerase to transcribe all of their genes. In E. coli, the polymerase is composed of five polypeptide subunits, two of which are identical. Four of these subunits, denoted α, α, β, and β’ comprise the polymerase core enzyme. These subunits assemble every time a gene is transcribed, and they disassemble once transcription is complete. Each subunit has a unique role; the two α-subunits are necessary to assemble the polymerase on the DNA; the β-subunit binds to the ribonucleoside triphosphate that will become part of the nascent “recently born” mRNA molecule; and the β’ binds the DNA template strand. The fifth subunit, σ, is involved only in transcription initiation. It confers transcriptional specificity such that the polymerase begins to synthesize mRNA from an appropriate initiation site. Without σ, the core enzyme would transcribe from random sites and would produce mRNA molecules that specified protein gibberish. The polymerase comprised of all five subunits is called the holoenzyme.

Question 32

How do prokaryotic promoters work?

Answer 32

In most cases, promoters exist upstream of the genes they regulate. The specific sequence of a promoter is very important because it determines whether the corresponding gene is transcribed all the time, some of the time, or infrequently. Although promoters vary among prokaryotic genomes, a few elements are conserved. At the -10 and -35 regions upstream of the initiation site, there are two promoter consensus sequences, or regions that are similar across all promoters and across various bacterial species. The -10 consensus sequence, called the -10 region, is TATAAT. The -35 sequence, TTGACA, is recognized and bound by σ. Once this interaction is made, the subunits of the core enzyme bind to the site. The A–T-rich -10 region facilitates unwinding of the DNA template, and several phosphodiester bonds are made. The transcription initiation phase ends with the production of abortive transcripts, which are polymers of approximately 10 nucleotides that are made and released.

Question 33

How does transcription elongation occur in prokaryotes?

Answer 33

The transcription elongation phase begins with the release of the σ subunit from the polymerase. The dissociation of σ allows the core enzyme to proceed along the DNA template, synthesizing mRNA in the 5’ to 3’ direction at a rate of approximately 40 nucleotides per second. As elongation proceeds, the DNA is continuously unwound ahead of the core enzyme and rewound behind it. The base pairing between DNA and RNA is not stable enough to maintain the stability of the mRNA synthesis components. Instead, the RNA polymerase acts as a stable linker between the DNA template and the nascent RNA strands to ensure that elongation is not interrupted prematurely.

Question 34

What are the kinds of termination signals for prokaryotic transcription?

Answer 34

Once a gene is transcribed, the prokaryotic polymerase needs to be instructed to dissociate from the DNA template and liberate the newly made mRNA. Depending on the gene being transcribed, there are two kinds of termination signals. One is protein-based and the other is RNA-based. Rho-dependent termination is controlled by the rho protein, Rho-independent termination is controlled by specific sequences in the DNA template strand.

Question 35

How does Rho-dependent termination work?

Answer 35

Rho-dependent termination is controlled by the rho protein, which tracks along behind the polymerase on the growing mRNA chain. Near the end of the gene, the polymerase encounters a run of G nucleotides on the DNA template and it stalls. As a result, the rho protein collides with the polymerase. The interaction with rho releases the mRNA from the transcription bubble.

Question 36

How does Rho-independent termination work?

Answer 36

Rho-independent termination is controlled by specific sequences in the DNA template strand. As the polymerase nears the end of the gene being transcribed, it encounters a region rich in C–G nucleotides. The mRNA folds back on itself, and the complementary C–G nucleotides bind together. The result is a stable hairpin that causes the polymerase to stall as soon as it begins to transcribe a region rich in A–T nucleotides. The complementary U–A region of the mRNA transcript forms only a weak interaction with the template DNA. This, coupled with the stalled polymerase, induces enough instability for the core enzyme to break away and liberate the new mRNA transcript.

Question 37

What happens on completion of transcription in prokaryotes?

Answer 37

Upon termination, the process of transcription is complete. By the time termination occurs, the prokaryotic transcript would already have been used to begin synthesis of numerous copies of the encoded protein because these processes can occur concurrently. The unification of transcription, translation, and even mRNA degradation is possible because all of these processes occur in the same 5’ to 3’ direction, and because there is no membranous compartmentalization in the prokaryotic cell. In contrast, the presence of a nucleus in eukaryotic cells precludes simultaneous transcription and translation.

Question 38

What is a CAAT box?

Answer 38

(GGCCAATCT) an essential eukaryotic promoter sequence involved in binding transcription factors.

Question 39

What is FACT?

Answer 39

Complex that “FAcilitates Chromatin Transcription” by disassembling nucleosomes ahead of a transcribing RNA polymerase II and reassembling them after the polymerase passes by.

Question 40

What is a GC-rich box?

Answer 40

(GGCG) a nonessential eukaryotic promoter sequence that binds cellular factors to increase the efficiency of transcription; may be present several times in a promoter.

Question 41

What is an Octamer box?

Answer 41

(ATTTGCAT) a nonessential eukaryotic promoter sequence that binds cellular factors to increase the efficiency of transcription; may be present several times in a promoter.

Question 42

What is a preinitiation complex?

Answer 42

A cluster of transcription factors and other proteins that recruit RNA polymerase II for transcription of a DNA template.

Question 43

What is small nuclear RNA (snRNA)?

Answer 43

Molecules synthesized by either RNA polymerase II or III that serve a variety of functions, including splicing pre-mRNAs and regulating transcription factors.

Question 44

How does eukaryotic transcription differ from prokaryotic transcription?

Answer 44

Prokaryotes and eukaryotes perform fundamentally the same process of transcription, with a few key differences. The most important difference between prokaryotes and eukaryotes is the latter’s membrane-bound nucleus and organelles. With the genes bound in a nucleus, the eukaryotic cell must be able to transport its mRNA to the cytoplasm and must protect its mRNA from degrading before it is translated. Eukaryotes also employ three different polymerases that each transcribe a different subset of genes. Eukaryotic mRNAs are usually monogenic, meaning that they specify a single protein.

Question 45

What is required for a eukaryotic RNA polymerase to bind to a DNA template?

Answer 45

Unlike the prokaryotic polymerase that can bind to a DNA template on its own, eukaryotes require several other proteins, called transcription factors, to first bind to the promoter region and then help recruit the appropriate polymerase.

Question 46

What does RNA polymerase I do?

Answer 46

RNA polymerase I is located in the nucleolus and synthesizes all of the rRNAs except for the 5S rRNA molecule.

Question 47

What is the nucleolus?

Answer 47

A specialized nuclear substructure in which ribosomal RNA (rRNA) is transcribed, processed, and assembled into ribosomes.

Question 48

What does rRNA do?

Answer 48

rRNA molecules are considered structural RNAs because they have a cellular role but are not translated into protein. The rRNAs are components of the ribosome and are essential to the process of translation.

Question 49

What is a Svedberg unit?

Answer 49

A nonadditive value that characterizes the speed at which a particle sediments during centrifugation.

Question 50

What does RNA polymerase II do?

Answer 50

RNA polymerase II is located in the nucleus and synthesizes all protein-coding nuclear pre-mRNAs. Eukaryotic pre-mRNAs undergo extensive processing after transcription but before translation. RNA polymerase II is responsible for transcribing the overwhelming majority of eukaryotic genes.

Question 51

What does RNA polymerase III do?

Answer 51

RNA polymerase III is also located in the nucleus. This polymerase transcribes a variety of structural RNAs that includes the 5S pre-rRNA, transfer pre-RNAs (pre-tRNAs), and small nuclear pre-RNAs.

Question 52

How can the polymerase that transcribes a eukaryotic gene be determined?

Answer 52

A scientist characterizing a new gene can determine which polymerase transcribes it by testing whether the gene is expressed in the presence of a particular mushroom poison, α-amanitin. Interestingly, α-amanitin produced by Amanita phalloides, the Death Cap mushroom, affects the three polymerases very differently. RNA polymerase I is completely insensitive to α-amanitin, meaning that the polymerase can transcribe DNA in vitro in the presence of this poison. In contrast, RNA polymerase II is extremely sensitive to α-amanitin, and RNA polymerase III is moderately sensitive. Knowing the transcribing polymerase can clue a researcher into the general function of the gene being studied.

Question 53

What is an example of a TATA box in eukaryotes?

Answer 53

In the mouse thymidine kinase gene, the TATA box is located at approximately -30 relative to the +1 site. For this gene, the exact TATA box sequence is TATAAAA, as read in the 5’ to 3’ direction on the non-template strand. This sequence is not identical to the E. coli TATA box, but it conserves the A–T rich element.

Question 54

How does the TATA box help promote transcription?

Answer 54

The thermostability of A–T bonds is low and this helps the DNA template to locally unwind in preparation for transcription.

Question 55

What is an example of a CAAT box?

Answer 55

The mouse genome includes one gene and two pseudogenes for cytoplasmic thymidine kinase. These pseudogenes are copied from mRNA and incorporated into the chromosome. For example, the mouse thymidine kinase promoter also has a conserved CAAT box (GGCCAATCT) at approximately -80.

Question 56

What is a pseudogene?

Answer 56

Genes that have lost their protein-coding ability or are no longer expressed by the cell.

Question 57

What nonessential promoter sequences can be found in eukaryotes that promote the efficiency of transcription?

Answer 57

Further upstream of the TATA box, eukaryotic promoters may also contain one or more GC-rich boxes (GGCG) or octamer boxes (ATTTGCAT). These elements bind cellular factors that increase the efficiency of transcription initiation and are often identified in more “active” genes that are constantly being expressed by the cell.

Question 58

What other molecules are involved in eukaryotic transcription other than polymerases and promoters?

Answer 58

Many basal transcription factors, enhancers, and silencers also help to regulate the frequency with which pre-mRNA is synthesized from a gene. Enhancers and silencers affect the efficiency of transcription but are not necessary for transcription to proceed. Basal transcription factors are crucial in the formation of a preinitiation complex on the DNA template that subsequently recruits RNA polymerase II for transcription initiation.

Question 59

What do basal transcription factors do?

Answer 59

The names of the basal transcription factors begin with “TFII” (this is the transcription factor for RNA polymerase II) and are specified with the letters A–J. The transcription factors systematically fall into place on the DNA template, with each one further stabilizing the preinitiation complex and contributing to the recruitment of RNA polymerase II.

Question 60

What is the difficulty in identifying eukaryotic promoter sequences?

Answer 60

It is difficult to infer exactly where a eukaryotic promoter begins and ends. Some promoters occur within genes; others are located very far upstream, or even downstream, of the genes they are regulating.

Question 61

What is the rate of evolution of eukaryotic promoters?

Answer 61

When researchers limited their examination to human core promoter sequences that were defined experimentally as sequences that bind the preinitiation complex, they found that promoters evolve even faster than protein-coding genes.

Question 62

Where are eukaryotic promoter sequences located?

Answer 62

In eukaryotes, the conserved promoter elements differ for genes transcribed by RNA polymerases I, II, and III. RNA polymerase I transcribes genes that have two GC-rich promoter sequences in the -45 to +20 region. These sequences alone are sufficient for transcription initiation to occur, but promoters with additional sequences in the region from -180 to -105 upstream of the initiation site will further enhance initiation. Genes that are transcribed by RNA polymerase III have upstream promoters or promoters that occur within the genes themselves.

Question 63

What happens after formation of the preinitiation complex?

Answer 63

Following the formation of the preinitiation complex, the polymerase is released from the other transcription factors, and elongation is allowed to proceed as it does in prokaryotes with the polymerase synthesizing pre-mRNA in the 5’ to 3’ direction. RNA polymerase II transcribes the major share of eukaryotic genes.

Question 64

How is eukaryotic DNA organized?

Answer 64

Although the enzymatic process of elongation is essentially the same in eukaryotes and prokaryotes, the DNA template is more complex. When eukaryotic cells are not dividing, their genes exist as a diffuse mass of DNA and proteins called chromatin. The DNA is tightly packaged around charged histone proteins at repeated intervals. These DNA–histone complexes, collectively called nucleosomes, are regularly spaced and include 146 nucleotides of DNA wound around eight histones like thread around a spool.

Question 65

How is transcription terminated in eukaryotes?

Answer 65

The termination of transcription is different for the different polymerases. Unlike in prokaryotes, elongation by RNA polymerase II in eukaryotes takes place 1,000–2,000 nucleotides beyond the end of the gene being transcribed. This pre-mRNA tail is subsequently removed by cleavage during mRNA processing. On the other hand, RNA polymerases I and III require termination signals. Genes transcribed by RNA polymerase I contain a specific 18-nucleotide sequence that is recognized by a termination protein. The process of termination in RNA polymerase III involves an mRNA hairpin similar to rho-independent termination of transcription in prokaryotes.

Question 66

What is a 7-methylguanosine cap?

Answer 66

A modification added to the 5’ end of pre-mRNAs to protect mRNA from degradation and assist translation.

Question 67

What is an anticodon?

Answer 67

A three-nucleotide sequence in a tRNA molecule that corresponds to an mRNA codon.

Question 68

What is an exon?

Answer 68

A sequence present in protein-coding mRNA after completion of pre-mRNA splicing.

Question 69

What is an intron?

Answer 69

A non-protein-coding intervening sequence that is spliced from mRNA during processing.

Question 70

What is a poly-A tail?

Answer 70

Modification added to the 3’ end of pre-mRNAs to protect mRNA from degradation and assist mRNA export from the nucleus.

Question 71

What is RNA editing?

Answer 71

Direct alteration of one or more nucleotides in an mRNA that has already been synthesized.

Question 72

What is splicing?

Answer 72

The process of removing introns and reconnecting exons in a pre-mRNA.

Question 73

What is the lifespan of mature eukaryotic mRNA compared to prokaryotic mRNA?

Answer 73

The additional steps involved in eukaryotic mRNA maturation create a molecule with a much longer half-life than a prokaryotic mRNA. Eukaryotic mRNAs last for several hours, whereas the typical E. coli mRNA lasts no more than five seconds.

Question 74

What happens during eukaryotic pre-mRNA processing?

Answer 74

Pre-mRNAs are first coated in RNA-stabilizing proteins; these protect the pre-mRNA from degradation while it is processed and exported out of the nucleus. The three most important steps of pre-mRNA processing are the addition of stabilizing and signaling factors at the 5’ and 3’ ends of the molecule, and the removal of intervening sequences that do not specify the appropriate amino acids. In rare cases, the mRNA transcript can be “edited” after it is transcribed.

Question 75

What is an example of organisms whose pre-mRNA must be edited in order to become functional?

Answer 75

The trypanosomes are a group of protozoa that include the pathogen Trypanosoma brucei, which causes sleeping sickness in humans. The mitochondrial DNA of trypanosomes exhibit an interesting exception to The Central Dogma: their pre-mRNAs do not have the correct information to specify a functional protein. Usually, this is because the mRNA is missing several U nucleotides. The cell performs an additional RNA processing step called RNA editing to remedy this.

Question 76

How is RNA editing performed in trypanosomes?

Answer 76

Other genes in the mitochondrial genome encode 40- to 80-nucleotide guide RNAs. One or more of these molecules interacts by complementary base pairing with some of the nucleotides in the pre-mRNA transcript. However, the guide RNA has more A nucleotides than the pre-mRNA has U nucleotides to bind with. In these regions, the guide RNA loops out. The 3’ ends of guide RNAs have a long poly-U tail, and these U bases are inserted in regions of the pre-mRNA transcript at which the guide RNAs are looped. This process is entirely mediated by RNA molecules. That is, guide RNAs—rather than proteins—serve as the catalysts in RNA editing.

Question 77

In which organisms does RNA editing occur?

Answer 77

RNA editing is not just a phenomenon of trypanosomes. In the mitochondria of some plants, almost all pre-mRNAs are edited. RNA editing has also been identified in mammals such as rats, rabbits, and even humans.

Question 78

What is the evolutionary explanation for RNA editing?

Answer 78

One possibility is that the mitochondria, being remnants of ancient prokaryotes, have an equally ancient RNA-based method for regulating gene expression. In support of this hypothesis, edits made to pre-mRNAs differ depending on cellular conditions. Although speculative, the process of RNA editing may be a holdover from a primordial time when RNA molecules, instead of proteins, were responsible for catalyzing reactions.

Question 79

How is the 5’ cap added to eukaryotic mRNA and what does it do?

Answer 79

While the pre-mRNA is still being synthesized, a 7-methylguanosine cap is added to the 5’ end of the growing transcript by a phosphate linkage. This moiety (functional group) protects the nascent mRNA from degradation. In addition, factors involved in protein synthesis recognize the cap to help initiate translation by ribosomes.

Question 80

How is the 3’ poly-A tail added to eukaryotic mRNA and what does it do?

Answer 80

Once elongation is complete, the pre-mRNA is cleaved by an endonuclease between an AAUAAA consensus sequence and a GU-rich sequence, leaving the AAUAAA sequence on the pre-mRNA. An enzyme called poly-A polymerase then adds a string of approximately 200 A residues, called the poly-A tail. This modification further protects the pre-mRNA from degradation and signals the export of the cellular factors that the transcript needs to the cytoplasm.

Question 81

Why do eukaryotic genes contain introns?

Answer 81

The discovery of introns came as a surprise to researchers in the 1970s who expected that pre-mRNAs would specify protein sequences without further processing, as they had observed in prokaryotes. The genes of higher eukaryotes very often contain one or more introns. These regions may correspond to regulatory sequences; however, the biological significance of having many introns or having very long introns in a gene is unclear. It is possible that introns slow down gene expression because it takes longer to transcribe pre-mRNAs with lots of introns. Alternatively, introns may be nonfunctional sequence remnants left over from the fusion of ancient genes throughout evolution. This is supported by the fact that separate exons often encode separate protein subunits or domains. For the most part, the sequences of introns can be mutated without ultimately affecting the protein product.

Question 82

How is splicing performed on pre-mRNAs?

Answer 82

All of a pre-mRNA’s introns must be completely and precisely removed before protein synthesis. If the process errs by even a single nucleotide, the reading frame of the rejoined exons would shift, and the resulting protein would be dysfunctional. Introns are removed and degraded while the pre-mRNA is still in the nucleus. Splicing occurs by a sequence-specific mechanism that ensures introns will be removed and exons rejoined with the accuracy and precision of a single nucleotide. The splicing of pre-mRNAs is conducted by complexes of proteins and RNA molecules called spliceosomes.

Question 83

How are tRNAs and rRNAs processed?

Answer 83

Most of the tRNAs and rRNAs in eukaryotes and prokaryotes are first transcribed as a long precursor molecule that spans multiple rRNAs or tRNAs. Enzymes then cleave the precursors into subunits corresponding to each structural RNA. Some of the bases of pre-rRNAs are methylated; that is, a –CH₃ moiety (methyl functional group) is added for stability. Pre-tRNA molecules also undergo methylation. As with pre-mRNAs, subunit excision occurs in eukaryotic pre-RNAs destined to become tRNAs or rRNAs.

Question 84

What do mature tRNAs and rRNAs do?

Answer 84

Mature rRNAs make up approximately 50 percent of each ribosome. Some of a ribosome’s RNA molecules are purely structural, whereas others have catalytic or binding activities. Mature tRNAs take on a three-dimensional structure through intramolecular hydrogen bonding to position the amino acid binding site at one end and the anticodon at the other end.

Question 85

What is aminoacyl tRNA synthetase?

Answer 85

An enzyme that “charges” tRNA molecules by catalyzing a bond between the tRNA and a corresponding amino acid.

Question 86

What is initiator tRNA?

Answer 86

In prokaryotes, called tRNA_f^Met; in eukaryotes, called tRNAⁱ; a tRNA that interacts with a start codon, binds directly to the ribosome P site, and links to a special methionine to begin a polypeptide chain.

Question 87

What are Kozak’s rules?

Answer 87

Rules that determine the correct initiation AUG in a eukaryotic mRNA; the following consensus sequence must appear around the AUG: 5’-GCC(purine)CCAUGG-3’; the bolded bases are most important.

Question 88

What is peptidyl transferase?

Answer 88

An RNA-based enzyme that is integrated into the large ribosomal subunit and catalyzes the formation of peptide bonds.

Question 89

What is a polysome?

Answer 89

An mRNA molecule simultaneously being translated by many ribosomes all going in the same direction.

Question 90

What is the Shine-Dalgarno sequence?

Answer 90

(AGGAGG); initiates prokaryotic translation by interacting with rRNA molecules comprising the 30S ribosome.

Question 91

What is a signal sequence?

Answer 91

A short tail of amino acids that directs a protein to a specific cellular compartment.

Question 92

What is a start codon?

Answer 92

AUG (or rarely, GUG) on an mRNA from which translation begins; always specifies methionine

Question 93

How much protein synthesis occurs in a cell?

Answer 93

The synthesis of proteins consumes more of a cell’s energy than any other metabolic process. In turn, proteins account for more mass than any other component of living organisms (with the exception of water), and proteins perform virtually every function of a cell.

Question 94

What is translation?

Answer 94

The process of translation, or protein synthesis, involves the decoding of an mRNA message into a polypeptide product. Amino acids are covalently strung together by interlinking peptide bonds in lengths ranging from approximately 50 amino acid residues to more than 1,000. Each individual amino acid has an amino group (NH₂) and a carboxyl (COOH) group. Polypeptides are formed when the amino group of one amino acid forms an amide (i.e., peptide) bond with the carboxyl group of another amino acid. This reaction is catalyzed by ribosomes and generates one water molecule.

Question 95

What are the components required for translation?

Answer 95

The composition of each component may vary across species; for instance, ribosomes may consist of different numbers of rRNAs and polypeptides depending on the organism. However, the general structures and functions of the protein synthesis machinery are comparable from bacteria to human cells. Translation requires the input of an mRNA template, ribosomes, tRNAs, and various enzymatic factors.

Question 96

How many ribosomes can be found in a cell?

Answer 96

In E. coli, there are between 10,000 and 70,000 ribosomes present in each cell at any given time.

Question 97

What is a ribosome composed of?

Answer 97

A ribosome is a complex macromolecule composed of structural and catalytic rRNAs, and many distinct polypeptides.

Question 98

Where can ribosomes be found?

Answer 98

Ribosomes exist in the cytoplasm in prokaryotes and in the cytoplasm and rough endoplasmic reticulum in eukaryotes. Mitochondria and chloroplasts also have their own ribosomes in the matrix and stroma, which look more similar to prokaryotic ribosomes (and have similar drug sensitivities) than the ribosomes just outside their outer membranes in the cytoplasm.

Question 99

What are the ribosomal subunits?

Answer 99

Ribosomes dissociate into large and small subunits when they are not synthesizing proteins and reassociate during the initiation of translation. In E. coli, the small subunit is described as 30S, and the large subunit is 50S, for a total of 70S (recall that Svedberg units are not additive). Mammalian ribosomes have a small 40S subunit and a large 60S subunit, for a total of 80S. The small subunit is responsible for binding the mRNA template, whereas the large subunit sequentially binds tRNAs.

Question 100

How is translation efficiency increased?

Answer 100

Each mRNA molecule is simultaneously translated by many ribosomes, all 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.

Question 101

What do tRNAs do?

Answer 101

The tRNAs are structural RNA molecules that were transcribed from genes by RNA polymerase III. 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.

Question 102

What are the factors that tRNAs need to interact with?

Answer 102

tRNAs need to interact with three factors: 1) they must be recognized by the correct aminoacyl synthetase; 2) they must be recognized by ribosomes; and 3) they must bind to the correct sequence in mRNA.

Question 103

How are amino acids attached to tRNAs?

Answer 103

Through the process of tRNA “charging,” each tRNA molecule is linked to its correct amino acid by a group of enzymes called aminoacyl tRNA synthetases. At least one type of aminoacyl tRNA synthetase exists for each of the 20 amino acids; the exact number of aminoacyl tRNA synthetases varies by species. These enzymes first bind and hydrolyze ATP to catalyze a high-energy bond between an amino acid and adenosine monophosphate (AMP); a pyrophosphate molecule is expelled in this reaction. The activated amino acid is then transferred to the tRNA, and AMP is released.

Question 104

What are the phases of translation?

Answer 104

As with mRNA synthesis, protein synthesis can be divided into three phases: initiation, elongation, and termination. The process of translation is similar in prokaryotes and eukaryotes.

Question 105

What is the translation initiation complex in prokaryotes made of?

Answer 105

In E. coli, this complex involves the small 30S ribosome, the mRNA template, three initiation factors (IFs; IF-1, IF-2, and IF-3), and a special initiator tRNA, called tRNA_f^Met.

Question 106

What does the initiator tRNA do in prokaryotes?

Answer 106

The initiator tRNA interacts with the start codon AUG (or rarely, GUG), links to a formylated methionine called fMet, and can also bind IF-2. Formylated methionine is inserted by fMet − tRNA_f^Met at the beginning of every polypeptide chain synthesized by E. coli, but it is usually clipped off after translation is complete. When an in-frame AUG is encountered during translation elongation, a non-formylated methionine is inserted by a regular Met-tRNA^Met.

Question 107

What does the Shine-Dalgarno sequence do?

Answer 107

In E. coli mRNA, a sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (AGGAGG), interacts with the rRNA molecules that compose the ribosome. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template.

Question 108

What is the energy source used during translation?

Answer 108

Guanosine triphosphate (GTP), which is a purine nucleotide triphosphate, acts as an energy source during translation—both at the start of elongation and during the ribosome’s translocation.

Question 109

What is the translation initiation complex in eukaryotes made of?

Answer 109

In eukaryotes, a similar initiation complex forms, comprising mRNA, the 40S small ribosomal subunit, IFs, and nucleoside triphosphates (GTP and ATP). The charged initiator tRNA, called Met-tRNA_i, does not bind fMet in eukaryotes, but is distinct from other Met-tRNAs in that it can bind IFs.

Question 110

How is the 5’ cap used by the eukaryotic initiation complex for translation?

Answer 110

Instead of depositing 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) and several other IFs assist 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.

Question 111

How does the eukaryotic initiation complex find the correct start codon?

Answer 111

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 either A or G, but cannot be C or U. Essentially, the closer the sequence is to this consensus, the higher the efficiency of translation.

Question 112

What is the final step of eukaryotic translation initiation?

Answer 112

Once the appropriate AUG is identified, the other proteins and CBP dissociate, and the 60S subunit binds to the complex of Met-tRNA_i, mRNA, and the 40S subunit. This step completes the initiation of translation in eukaryotes.

Question 113

What are the sites of the large ribosomal subunit that are used during translation?

Answer 113

In prokaryotes and eukaryotes, the basics of elongation are the same. The 50S ribosomal subunit of E. coli consists of three compartments: the A (aminoacyl) site binds incoming charged aminoacyl tRNAs. The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E (exit) site releases dissociated tRNAs so that they can be recharged with free amino acids. There is one exception to this assembly line of tRNAs: in E. coli, fMet−tRNA_f^Met is capable of entering the P site directly without first entering the A site. Similarly, the eukaryotic Met-tRNA_i, with help from other proteins of the initiation complex, binds directly to the P site. In both cases, this creates an initiation complex with a free A site ready to accept the tRNA corresponding to the first codon after the AUG.

Question 114

How is specificity provided for tRNAs during translation?

Answer 114

During translation elongation, the mRNA template provides specificity. As the ribosome moves along the mRNA, each mRNA codon comes into register, 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.

Question 115

How does elongation occur during translation?

Answer 115

Elongation proceeds with charged tRNAs entering the A site and then shifting to the P site followed by the E site with each single-codon “step” of the ribosome. Ribosomal steps are induced by conformational changes that advance the ribosome by three bases in the 3’ direction. The energy for each step of the ribosome is donated by an elongation factor that hydrolyzes GTP. Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA. The formation of each peptide bond is catalyzed by peptidyl transferase, an RNA-based enzyme that is integrated into the 50S ribosomal subunit. The energy for each peptide bond formation is derived from GTP hydrolysis, which is catalyzed by a separate elongation factor. The amino acid bound to the P-site tRNA is also linked to the growing polypeptide chain. As the ribosome steps across the mRNA, the former P-site tRNA enters the E site, detaches from the amino acid, and is expelled.

Question 116

How fast is translation?

Answer 116

Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200-amino acid protein can be translated in just 10 seconds.

Question 117

How is translation terminated?

Answer 117

Termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered. Upon aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes and eukaryotes that instruct peptidyl transferase to add a water molecule to the carboxyl end of the P-site amino acid. This reaction forces the P-site amino acid to detach from its tRNA, and the newly made protein is released. The small and large ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation initiation complex. After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused in another transcription reaction.

Question 118

What happens after translation?

Answer 118

During and after translation, individual amino acids may be chemically modified, signal sequences may be appended, and the new protein “folds” into a distinct three-dimensional structure as a result of intramolecular interactions. A signal sequence is added to the amino end or the carboxyl end of the protein. Other cellular factors recognize each signal sequence and help transport the protein from the cytoplasm to its correct compartment. Once the protein reaches its cellular destination, the signal sequence is usually clipped off.

Question 119

How do chaperones help in protein folding?

Answer 119

Many proteins fold spontaneously, but some proteins require helper molecules, called chaperones, to prevent them from aggregating during the complicated process of folding. Even if a protein is properly specified by its corresponding mRNA, it could take on a completely dysfunctional shape if abnormal temperature or pH conditions prevent it from folding correctly.