Sitemap
|
|
|
|
|
Now, let's now turn our attention to protein synthesis, or more properly, polypeptide synthesis. The amino acid sequence of a protein is encoded within the DNA. The sequence of events, starting with the transcription of messenger RNA and terminating with the translation of mRNA information into polypeptide product, is called gene expression. Key words for this section are: transcription, RNA polymerase, processing, intron, exon, codon, anticodon, and translation.
Polypeptide synthesis requires three different classes of RNA, all of which are encoded in the DNA. When a DNA gene is read, a particular RNA species is produced. Often, these RNA species play one of several critical roles in the process of translation. Recall that DNA is double stranded and that the two strands are complementary. That is, the sequence of bases on one strand depends upon the sequence on the other strand. This is because bases hydrogen bond with each other and their structure permits only certain pairings for bonding. Thymine, T, on one strand can pair with adenine, A, on the other strand. This pair forms two hydrogen bonds. Cytosine, C, on one strand pairs with guanine, G, on the other. The G/C pair forms three hydrogen bonds.
So, if one strand of DNA reads C-C-C-T-T-T, the other complementary strand would read G-G-G-A-A-A. Each strand of DNA has a 5-prime (5') end and a 3-prime (3') end. These designations refer to particular carbon atoms in the deoxyribose sugars that, along with phosphate groups, form the backbone of a DNA strand. Let's analogize deoxyribose to a flag pole. The slender top is the 5' end and the thickened pedestal or base is the 3' end. The ball at the top of the pole is a phosphate group and the flag can be the base. If we stacked poles one on top of another, base to ball, we'd produce a long chain. At one end would be a ball; at the far end would be a pedestal or base.
So it is with DNA, at the 5' end of a strand, there is a phosphate group attached to the number five carbon of deoxyribose of the terminal nucleotide, and the strand stops there. At the other end of the strand, the 3' end, the terminal nucleotide has a deoxyribose with its 3' carbon exposed, and the strand stops there. RNA, which is single stranded, has the same kind of directionality, with 5' and 3' ends. When two strands of nucleic acid hydrogen bond, they are each oriented in opposite directions; the 5' end of one is next to the 3' end of the other. They are said to be antiparaliel.
Transcription is the process of making an RNA species as a complement to one of the DNA strands. Only a relatively tiny proportion of a DNA strand is ever used to direct the synthesis of any given RNA. The three main classes of RNA that must be transcribed for protein synthesis to occur are ribosomal or rRNAs, transfer or tRNAs, and messenger or mRNAs. Within each class of RNA there are different particular species or examples. In eukaryotes, there are four kinds of rRNAs, 32 kinds of tRNAs, and thousands of mRNAs, one per kind of polypeptide produced by the cell. rRNAs provide the physical plant for protein synthesis. One each of the several RRNA varieties associate along with dozens of proteins to produce a ribosome. Protein synthesis occurs on ribosomes in the cytoplasm.
The ribosome attaches to the 5' end of mRNA. It takes energy to move the ribosome down the message.
Protein Synthesis
Now, let's now turn our attention to protein synthesis, or more properly, polypeptide synthesis. The amino acid sequence of a protein is encoded within the DNA. The sequence of events, starting with the transcription of messenger RNA and terminating with the translation of mRNA information into polypeptide product, is called gene expression. Key words for this section are: transcription, RNA polymerase, processing, intron, exon, codon, anticodon, and translation.
Polypeptide synthesis requires three different classes of RNA, all of which are encoded in the DNA. When a DNA gene is read, a particular RNA species is produced. Often, these RNA species play one of several critical roles in the process of translation. Recall that DNA is double stranded and that the two strands are complementary. That is, the sequence of bases on one strand depends upon the sequence on the other strand. This is because bases hydrogen bond with each other and their structure permits only certain pairings for bonding. Thymine, T, on one strand can pair with adenine, A, on the other strand. This pair forms two hydrogen bonds. Cytosine, C, on one strand pairs with guanine, G, on the other. The G/C pair forms three hydrogen bonds.
So, if one strand of DNA reads C-C-C-T-T-T, the other complementary strand would read G-G-G-A-A-A. Each strand of DNA has a 5-prime (5') end and a 3-prime (3') end. These designations refer to particular carbon atoms in the deoxyribose sugars that, along with phosphate groups, form the backbone of a DNA strand. Let's analogize deoxyribose to a flag pole. The slender top is the 5' end and the thickened pedestal or base is the 3' end. The ball at the top of the pole is a phosphate group and the flag can be the base. If we stacked poles one on top of another, base to ball, we'd produce a long chain. At one end would be a ball; at the far end would be a pedestal or base.
So it is with DNA, at the 5' end of a strand, there is a phosphate group attached to the number five carbon of deoxyribose of the terminal nucleotide, and the strand stops there. At the other end of the strand, the 3' end, the terminal nucleotide has a deoxyribose with its 3' carbon exposed, and the strand stops there. RNA, which is single stranded, has the same kind of directionality, with 5' and 3' ends. When two strands of nucleic acid hydrogen bond, they are each oriented in opposite directions; the 5' end of one is next to the 3' end of the other. They are said to be antiparaliel.
Transcription is the process of making an RNA species as a complement to one of the DNA strands. Only a relatively tiny proportion of a DNA strand is ever used to direct the synthesis of any given RNA. The three main classes of RNA that must be transcribed for protein synthesis to occur are ribosomal or rRNAs, transfer or tRNAs, and messenger or mRNAs. Within each class of RNA there are different particular species or examples. In eukaryotes, there are four kinds of rRNAs, 32 kinds of tRNAs, and thousands of mRNAs, one per kind of polypeptide produced by the cell. rRNAs provide the physical plant for protein synthesis. One each of the several RRNA varieties associate along with dozens of proteins to produce a ribosome. Protein synthesis occurs on ribosomes in the cytoplasm.
The ribosome attaches to the 5' end of mRNA. It takes energy to move the ribosome down the message. The ribosome moves toward the 3' end of the message three bases at a time. Each set of three bases specifies a particular amino acid. At the ribosome, complementary base pairing occurs between amino acid-carrying tRNAs and the message. This ensures that amino acids carried by tRNAs arrive in the order specified by the sequence of three base sets on the mRNA. Ribosomal enzymes catalyze the attachment of each newly arrived amino acid to the growing polypeptide chain.
mRNA carries, in its sequence of three-base sets, the information for the sequence of amino acid residues of a polypeptide. Each set of three bases is called a codon. There are 61 different codons used to specify amino acids. But, because there are only 20 different amino acids used in translation, some amino acids are encoded by several different codons. This situation is called codon synonymy.
A tRNA binds to mRNA by complementarily base pairing with a codon. The three bases of the tRNA that form the hydrogen bonds with the mRNA codon are called the anticodon. Because there are only 32 kinds of tRNAs and, correspondingly, 32 anticodons in eukaryotes but 61 kinds of codons; some anticodons must pair with multiple codons.
Some sloppiness, then, is tolerated in complementary base pairing between mRNA and tRNA. This sloppiness is called wobble and always involves the third, or 3', base of the codon.
There are 32 kinds of tRNA but only 20 kinds of amino acids used in translation. This means some amino acids are carried on more than one kind of tRNA. Ail especially important example of this involves the amino acid methionine. Methionine, in eukaryotes, and its derivative, formylmethionine, or f-met, in prokaryotes, is the first amino acid in the sequence as a polypeptide is being synthesized.
The initial methionine is carried on a specialized tRNA that recognizes the so called start codon, 5' A-U-G 3'. The tRNA that carries the initial methionine is more slender than a typical tRNA, allowing it to fit into a particular pocket of the ribosome, called the P site. At the start of translation, the P site lies above the start codon. If, later in the message, methionine is specified, it will be carried by a different tRNA capable of fitting into a different crevice or pocket of the ribosome, the A site. The A site always comes to lie above the next codon to be read as the ribosome is moved down the message.
We'll now consider some details of protein synthesis, then return to RNA transcript production in the nucleus. The ribosome is a two part structure. In eukaryotes, it is composed of over 80 proteins plus four ribosomal RNAs or rRNAs. The larger subunit contains 28S, 5.8S, and 5S rRNAs while the smaller subunit contains the 18S rRNA. Here, the term "S" refers to a unit, the Svedberg, which depends on both size and shape. When a message, mRNA, reaches the cytoplasm, it attaches to the small ribosome subunit which includes the 18S ribosomal RNA, then the large subunit attaches.
In prokaryotes, the message and ribosome attach by way of complementary base pairing between the 5' end of the message and the 3' end of the small subunit rRNA, a 16S species. In eukaryotes, an oddly oriented nucleotide at the 5' end of the message assists in binding the ribosome, without complementary base pairing. This oddly oriented nucleotide contains a modified base, 7-methylguanosine.
Once the ribosome is attached to the message, its P site sits over the first codon of the message. The first codon is not, however, the first three bases of the mRNA. 5' to the first, or start, codon are a small number of bases, or more precisely, nucleotides, that comprise the leader sequence. Leaders play a role in attachment of the ribosome to the message. With the P site over the start codon, the A site sits over the second codon.
The first tRNA to bind carries methionine, or f-met in prokaryotes. This tRNA is called the initiator aminoacyl-tRNA. The prefix aminoacyl implies that the tRNA is, in fact, carrying an amino acid. The initiator aminoacyl-tRNA binds to the message by codon-anticodon base pairing at the P site.
Next, another aminoacyl-tRNA moves into the A site and base pairs with the second codon. A ribosomal enzyme then catalyzes the formation of a peptide bond between the two amino acids carried by the two tRNAs. The tRNA in the A site now carries both amino acids. tRNAs carrying multiple amino acids are called peptidyl-tRNAs.
Amino acids are attached to tRNAs by their carboxyl, or -COOH, groups. In peptide bond formation, the carboxyl group of the amino acid at the P site is attached to the amino, or -NH3, group of the amino acid in the A site. Because of this, the amino acid of the tRNA in the P site is transferred to the tRNA in the A site. That leaves an empty tRNA in the P site and a peptidyi-tRNA in the A site. The empty tRNA then leaves the P site.
Next, the peptidyl-tRNA in the A site is moved to the P site. However, due to its size, the peptidyl tRNA does not fit down into the P site and does not contact the messenger RNA. Once the peptidvl-tRNA is translocated to the P site, the ribosome's A site is vacant. At this point, the whole ribosome slides down the messenger RNA by exactly one codon, or three bases. This means the vacant A site lies over the next, unread codon. Another aminoacyl-tRNA then base pairs with the message at the newly exposed codon and the process of peptide bond formation is repeated. That is, the peptide held by the tRNA at the P site is transferred to the aminoacyl-tRNA in the A site. The carboxyl end of the P site peptide is attached to the exposed amino end of the A site amino acid. The whole process of translocation from A to P site, ribosome movement down the message, and peptide bond formation is repeated unti I, at last, the A site I ies over a stop codon.
There are three different possible stop codons, none of which specify an amino acid. They are, from 5' to 3', U-G-A, U-A-A, and U-A-G. Because no tRNAs routinely bind to stop codons, translation usually halts at the stop codon. The newly formed polypeptide is then released from the peptidyl-tRNA at the A site by an enzyme. The empty tRNA leaves the A site, and the ribosome falls off of the message.
Just as the start codon is not at the very beginning of the message, the stop codon is not at the very end. A trailer sequence of bases follows the stop codon. In eukaryotes, a tail, up to several hundred bases long usually occurs at the 3' end of mRNA.
The message may be re-read or destroyed. Sometimes a single message is read simultaneously by several ribosomes. Collectively, these ribosomes are called polysomes. Here, as one ribosome moves down the message, another may attach.
During translation, ribosomal enzymes play important roles. Enzymes catalyze the translocation of peptidyl tRNAs from the A to P site, and the translocation of the ribosome down the message, a codon at a time. Both of these events require the hydrolysis or splitting of GTP into GDP plus phosphate. An ATP requiring event is the bonding of the amino acid to the tRNA to form an aminoacyl-tRNA.
Think of the attachment of the amino acid to the tRNA as a form of activation. ATP donates energy to activate or energize amino acids, making them more reactive. Amino acids not attached to tRNAs do not readily polymerize into polypeptides. The release of the polypeptide from the peptidyl-tRNA also requires ATP. Proteins called release factors, facilitate the release of the message by the ribosome.
Now, we'll discuss transcription, the process of producing an RNA molecule as a complement to one of the two strands of DNA. Each of the three classes of RNA is produced when DNA genes are read. Ribosomal RNAs come from the reading of RRNA genes. Transfer RNAs come from the reading of TRNA genes and messenger RNAs come from the reading of protein-specifying (structural) genes.
In eukaryotes, transcription occurs in the nucleus where the DNA Is confined. In prokaryotes, there is no nucleus. So, transcription occurs in the cytoplasm where the ribosomes of translation are also found. Consequently, in prokaryotes translation of mRNA may begin before transcription is even completed.
The enzyme that catalyzes RNA synthesis as a complement to a DNA strand is RNA polymerase. This enzyme must bind to the DNA and must also bind the RNA building blocks which are the four nucleoside triphosphates (= NTPS), ATP, GTP, CTP, and UTP. As the enzyme moves down a DNA strand it proceeds in the direction 3' to 5'. This, of course, results in production of the 5' end of the transcript first because the newly synthesized transcript and its complementary DNA strand will be oriented antiparallel to one another.
For example, if the DNA reads 3' T-A-C 5' on the strand the RNA polymerase is reading, the polymerase 'will bind and link together the nucleoside triphosphates ATP, UTP, and GTP. This would produce an RNA reading 5'A-U-G 3'. The RNA A-U-G is complementary to the DNA strand reading T-A-C. But, the RNA is produced antiparallel to the DNA. The RNA polymerase moves down the DNA towards its 5' end but it's the 5' end of the RNA that's made first.
When the RNA polymerase uses a nucleoside triphosphate to extend a growing RNA strand, the last two phosphates are removed. Consequently, the RNA backbone is simply alternating ribose sugars and phosphate groups.
Prokaryotes use a single RNA polymerase to synthesize all classes of RNA. But eukaryotes employ several different RNA polymerases. RNA Polymerase I catalyzes the synthesis of most of the ribosomal RNAS; RNA Polymerase 11 catalyzes messenger RNA synthesis; and RNA polymerase III catalyzes the synthesis of transfer RNAS, one kind of ribosomal RNA, and another group of RNAs called small nuclear RNAs or snRNAs.
When an RNA molecule has just been produced as a complement to one DNA strand, it is usually not yet ready to perform its function in translation. Rather, it must first be post-transcriptionally modified, where "post" means after. Post-transcriptional RNA modification is called RNA processing. Each of the three main classes of RNA, tRNA, mRNA, and rRNA requires a different kind of processing.
Let's begin with rRNA processing. In eukaryotes, three of the four genes for rRNAs are grouped together in the DNA as a single unit, but within the group each is separated from the others by spacer DNA. Also, several hundred copies of this unit of three rRNA genes lie together on the DNA. Each unit of three genes is separated from adjacent units by spacer DNA.
The spacer DNA, whether between adjacent sets of genes or between genes within a set, does not belong in the ribosome. Therefore, spacers must be enzymatically cleaved away from the transcription product before functional RNA species are produced. A single rRNA transcript contains the three rRNAs of a set, plus the spacers between them, plus some of the spacer that separates one set from another.
Genes for transfer RNAs also occur in multiple copies in the DNA. Sometimes they are clustered together and sometimes not. In either case, the transcript must be processed. First, the tri-nucleotide, 5' C-C-A 3', is added to one end (3') of the transfer RNA. The amino acid that the tRNA will eventually carry is covalently bonded to a ribose sugar of the A nucleotide of the C-C-A. Often, many of the bases of a tRNA transcript are chemically modified.
Sometimes the DNA corresponding to a tRNA gene contains a sequence that does not belong in the final RNA product. Complete processing requires that this sequence, called an intron, be enzymatically cleaved out of the transcript. Many genes in eukaryotes contain introns. Once these genes are transcribed, the introns must be removed to yield a mature, functional transcript.
Removal of introns is an important part of mRNA transcript processing in eukaryotes. In prokaryotes, protein genes do not contain introns. Consequentiy, their mRNAs are said to be colinear, meaning that their sequence of bases is exactly the sequence necessary to specify the order of amino acids in the protein product of translation. But in eukaryotes, initial or pre-mRNAs are not colinear. The sequence of codons is interrupted by introns. The introns are cleaved out of the pre-mRNA with the aid of small nuclear RNAs that bind to the introns. This makes the introns obvious to processing enzymes.
The parts of the gene or transcript that specify amino acids are called exons. When introns are removed, the exons are spliced together and the message becomes colinear. The role of introns in genes is unknown. Early in the origin of life, they may have served to sever the ties between coding sequences for functional domains of proteins. This might, then, have permitted mixing and matching of various exons to produce transcripts for a variety of proteins from a small number of encoded domains. Exon shuffling refers to the mixing and matching process.
Another common event in mRNA transcript processing is the addition of a chain of adenine containing nucleotides to the 3' end. This poly-A tail, as it is called, may be several hundred bases long. Its role Is not clear. However, transcripts with the tail often survive longer in the cytoplasm before they are degraded by ribonucleases.
Finally, in eukaryotes, the mRNA transcript is capped. Capping is the addition of a methylguanosine containing nucleotide to the 5' end of the transcript. This nucleotide, which is attached in an unusual manner, appears to aid in the attachment of the ribosome to the messenger RNA.
Let's now examine a eukaryote protein gene in a little more detail. We've already seen that the gene is not simply a sequence of bases that is complementary to the sequence of codons in mRNA. There may be one or more introns intervening between exons. Genes have other parts as well.
A gene contains one or two promoters. These are short sequences that serve as binding signals to RNA polymerase. The first of these is the "cat" box, so named because on average it contains the sequence C-A-A-T. Genes without CAAT boxes are transcribed only 1/50th as often as genes with them. Closer to the start point of transcription is another promoter called the "TATA" box because on average it contains the sequence T-A-T-A. The RNA polymerase binds to the DNA at this promoter and, once bound, overlaps the start point for transcription.
The first part of the gene to be transcribed is a leader sequence. This sequence may be up to several hundred bases long. It is neither exon nor intron. It is not removed during processing but neither does its base sequence encode or specify amino acids. The leader is followed by the initiation or start codon. This is followed by the rest of the first exon. Each set of three bases specifies or encodes a particular amino acid. The first exon may be followed by an intron, then another exon, then another intron, then another exon, etc. The end of the last exon contains three bases that in the mRNA will signal the stop point for translation. However, the gene does not stop here. Beyond the last exon is a trailer sequence which, at its end, contains the poly-A tail attachment site. Transcription proceeds to the end of the trailer sequence.
The gene, then, begins with promoters and ends with a trailer sequence. The part transcribed into RNA begins with the leader and ends with the trailer. As the transcript is processed, introns are excised and exons are spliced. Translation occurs along that part of the transcript from the initiation or start codon to the stop codon. Neither the leader nor the trailer of the transcript are translated. Only the base sequence of exons is translated. Each set of 3 bases along the exon base sequence corresponds to an amino acid-specifying codon.
Now, let's review the key words: transcription, RNA polymerase, processing, intron, exon, codon, anticodon, and translation.
Transcription is the process of synthesizing an RNA strand as a complement to one of the two strands of DNA. The final RNA product is very small compared to the length of a DNA molecule. A single DNA molecule contains the information necessary to specify a great many different RNAS.
RNA polymerases are the class of enzymes that catalyze RNA synthesis. These enzymes bind to a strand of DNA. They also bind the building blocks of RNA, nucleoside triphosphates. The building blocks are selected, one at a time, according to the DNA base with which they must complementarily pair. Each successive building block is then linked as a nucleotide to the 3' end of the growing RNA chain. RNA transcripts must be processed before they become functional.
Introns must be removed or excised from messenger and transfer RNA transcripts and the remaining exons must be spliced or covalently linked together. Introns are extraneous sequences devoid of useful information. Exons are those transcript sequences essential to the function of the final RNA product.
A messenger - or mRNA transcript carries a series of three-base sequences called codons. Each codon specifies a particular amino acid and the order of codons in the mRNA determines the order of amino acids in the protein specified by that mRNA.
An anticodon is a three-base sequence on a transfer - or tRNA that permits it to complementarily pair, or hydrogen bond, with a codon on the mRNA. Each transfer RNA carries only one type of amino acid, out of 20 possible types. The amino acid carried by a tRNA depends on its anticodon sequence.
Translation is the process of assembling a protein based on the codon order in messenger RNA. Ribosomes aid in the process by facilitating the binding of a tRNA to an mRNA. Ribosomes slide down the message, pausing at each codon. At each pause, a transfer RNA pairs with the message, anticodon to codon, thereby, delivering its amino acid. Each successive amino acid to be delivered is attached via a peptide bond to those having arrived previously. Enzymes of the ribosome catalyze peptide bond formation.
Top of Page
|
|
|
