Introduction

Protein biosynthesis can be divided into the steps of transcription, processing, and translation. During transcription, reading genetic information, encrypted in DNA molecules, and recording this information in mRNA molecules. During a series of successive stages of processing, some fragments that are unnecessary in subsequent stages are removed from mRNA, and nucleotide sequences are edited. After the code is transported from the nucleus to the ribosomes, the actual synthesis of protein molecules occurs by attaching individual amino acid residues to the growing polypeptide chain.

Processing

Between transcription and translation, the mRNA molecule undergoes a series of successive changes that ensure the maturation of a functioning template for the synthesis of the polypeptide chain. With the advent of processing in the eukaryotic cell, it became possible to combine gene exons to obtain a greater variety of proteins encoded by a single DNA nucleotide sequence.

capping

The chemical structure of the cap

During capping, the 7-methylguanosine transcript is attached to the 5'-end via a triphosphate bridge connecting them in an unusual position 5'-5', as well as methylation of the riboses of the first two nucleotides. The capping process begins even before the end of transcription of the pre-mRNA molecule.

Cap group functions:

regulation of mRNA export from the nucleus;
protection of the 5'-end of the transcript from exonucleases;
participation in the initiation of the broadcast

Polyadenylation

Polyadenylation consists in attaching from 100 to 200 adenylic acid residues to the 3' end of the transcript, carried out by a special enzyme poly(A) polymerase.

Splicing

After polyadenylation, the mRNA undergoes the removal of introns. The process is catalyzed by the spliceosome and is called splicing.

Broadcast

The finished protein molecule is then cleaved from the ribosome and transported to the right place in the cell. To achieve your active state some proteins require additional post-translational modification.

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This is a set of processes that ensure the transformation of synthesized RNA (RNA transcript) into functionally active RNA (mature RNA) that can be used in protein synthesis. The RNA transcripts themselves are not functionally active. The process is characteristic of eukaryotes.

As a result of processing, the structure and chemical organization of RNA changes. The RNA transcript prior to the formation of mature RNA is called pro-mRNA(or depending on the type of RNA - pro-tRNA, pro-rRNA), i.e. RNA precursor. Almost all RNA transcripts of eukaryotes and prokaryotes (excluding prokaryotic mRNA) are being processed. The transformation of an RNA transcript into mature RNA begins in the nucleus, when RNA synthesis has not yet been completed and it has not separated from DNA. Depending on the mechanisms, several stages of RNA maturation are distinguished.

Interaction of pro-mRNA with protein.

Methylation of pro-mRNA.

Capping the 5' end.

Polyadenylation.

Splicing.

The graphical sequence of stages is shown in Figure 58. It should be noted that in living organisms all of the above processes run parallel to each other.

A. Interaction of pro-mRNA with protein.

In bacteria, even before the end of transcription 5', the end of the transcript immediately connects to the ribosome and the mRNA is included in the translation. Therefore, practically no modification is required for bacterial mRNA. In eukaryotes, the synthesized transcript leaves the nucleus, enters the cytoplasm, and there it joins the ribosome. On its way, it must be protected from accidental encounters with strong reagents and, at the same time, be accessible to processing enzymes. Therefore, the RNA transcript immediately interacts with the protein as it is elongated. An analogy is appropriate here - the RNA transcript is located on the protein as on an operating table, it is fixed by chemical bonds, and at the same time modification sites become available in it. The RNA associated with a protein is called a ribonucleoprotein (informosome). In this form, the transcript is located in the nucleus. When leaving the nucleus, some RNAs continue to remain in combination with the protein, while others leave the complex and take part in translation.

b. Methylation of pro-mRNA.

Most often occurs in bacteria that have a special defense mechanism against foreign

DNA (viral, phage). This apparatus consists of a number of enzymes that cut foreign DNA or RNA at certain sites in which a specific nucleotide sequence is located. Enzymes are called restrictases. It is clear that one's own newly synthesized RNA transcript can also be attacked by restriction enzymes. To prevent this from happening, special enzymes called methylases, methylate their own RNA transcript at sites that can be cut by their own enzymes. In eukaryotes, the RNA transcript is less methylated.

Promoter Terminator

Transcription

Pro-mRNA fixi-Protein

torn on protein

Pro-mRNA methylation

Capping of pro-mRNA

Rice. 58. Scheme of the main points of processing.

V. Capping 5' end.

Consists of chemical and conformational change

5'end of the synthesized RNA. Capping occurs at the time of RNA synthesis, even before its separation. The process consists in attaching to the free end of the pro-RNA special chemical substances, which change the conformation of the terminal region. Capping is necessary to initiate the translation process.

Special enzymes attach to the 5'end of the pro-mRNA GDP (guanosine diphosphate) and then methylate it.

5' pro-mRNA

CH 3

CEP = HDF + CH 3

Fig.59. The structure of the CEP at the 5'end of eukaryotic pre-mRNA.

CEP functions.

Initiates protein synthesis.

Protects pro-mRNA from decay.

Involved in the removal of introns.

d. Polyadenylation.

This is the process of attaching 100-200 adenylic acid residues to the 3' end of the pro-mRNA. These residues are called poly-A sequences (poly-A tails). Not all pro-mRNAs undergo polyadenylation. For example, molecules of all types of histones do not contain poly-A sequences. Polyadenylation prevents mRNA from being destroyed.

The growing mRNA chain has a special nucleotide sequence (AAAAAA). A special enzyme (polyA polymerase) finds this combination of nucleotides, cuts the pro-mRNA in this place and forms a polyadenyl tail.

The value of poly-A sequences:

Facilitate the release of mRNA from the nucleus into the cytoplasm.

Protect mRNA from destruction.

Recently, another interesting poly-A property sequences - they are involved in the termination of pro-mRNA synthesis. RNA polymerase, forming the AAUAAAA sequence in pro-mRNA, receives a signal about the completion of the synthesis of the RNA transcript. But the synthesis does not stop immediately. It stops completely after RNA polymerase encounters a specific nucleotide sequence on the DNA template strand (it is different for different genes), which gives the final signal to stop RNA synthesis.

GTP PolyA - sequence

rArArArArArArArA-ON

CH 3

CEP = GTP + CH 3

Rice. 60. The structure of the CEP at the 5'end of eukaryotic pro-mRNA and the polyadenyl sequence at the 3'end of pro-mRNA.

e. Splicing.

IN The RNA transcript contains a certain number of nucleotide sequences that were necessary for the successful completion of translation and subsequent modification of the transcript (capping, polyadenylation, etc.). To perform the main role of RNA in the cytoplasm - translation, these sequences will not only have no functional significance, but may interfere with the normal course of protein synthesis. Therefore, the cell provides a mechanism for the release of the primary transcript from a number of sequences that are not critical in translation.

These sequences are primarily introns.

The gene from which pro-mRNA was transcribed contains coding and non-coding sequences. The coding sequences of a gene determine the amino acid and their sequence in the protein. Non-coding sequences do not have this property. The coding and non-coding sequences in a gene alternate, and their number depends on the individual genes. The primary transcript also contains coding and non-coding sequences. This organization of genes and pro-RNA is characteristic of eukaryotes. Non-coding pro-mRNA sequences are called introns, and the coding exons. The length of introns can be from 50 to 12,000 nucleotides. Gene starts and

ends with an exon. The discontinuous structure of the gene is characteristic of most eukaryotes. Introns can contain all types of RNA - mRNA, tRNA, rRNA.

The entire set of exons (coding proteins) in the human genome occupy only 1.1 - 1.4%. The average human gene contains 9 introns. As you simplify

organization of organisms, the total value of their exons increases (for example, in bacteria it is 86%).

A multicomponent complex takes part in the excision of introns from the RNA transcript and the fusion of the remaining exons. Its main components are small nuclear RNA (snRNA) and enzyme proteins.

In general, the complex is called small nuclear ribonucleoproteins, snRNPs orspliosome . The process itself is quite complicated and consists of several stages (see Fig. 58).

1. Shapingspliosomes . Fragments of protein and snRNA are attached to the beginning and end of the intron (Fig. 56, E) forming a spliosome. (Fig. 56, E) Attachment of the snRNP complex (Fig. 56, F).

Exon 1 Intron Exon 2

A loop

intron cut out

Rice. 61. Scheme of splicing (explanation in the text).

Approximation of neighboring exons, due to the formation of an intron loop. Cutting at the exon-intron boundary and connecting neighboring (first and second) exons (Fig. 56, C).

Removal and destruction of the loop and spliosome (Fig. 56, D, G).

It should be noted that if the intron is damaged (mutated), splicing may not be completed, the intron may not be excised, and the end product, mRNA, will carry nucleotide sequences that are unusual for it. It is clear that this can lead to disruption of translation and exclusion of a certain protein from the metabolism.

e. Alternative splicing.

This type of splicing occurs when the same gene is expressed in different tissues.

Its essence is that the same section of a gene in different tissues can act as an intron and an exon. This leads to the formation of different mRNAs that encode proteins with different enzymatic activities.

So in the cells of the thyroid gland, the hormone calcitonin is synthesized. It inhibits the release of calcium from bones. The gene that controls calcium synthesis

The gene that controls calcitonin

uh and uh and uh and uh and uh

1 2 3 4 5 6

uh and uh and uh and uh and uh

pro-mRNA

1 2 3 4 5 6

in the thyroid gland in the brain cells

mRNA

1 2 3 4 1 2 3 5 6

Calcitonin Calcitonin-like protein

Fig.62. Alternative splicing of calcitonin and calcitonin-like protein.

cytonin, consists of 6 exons, the primary transcript of this gene (pro-mRNA) also consists of 6 exons (Fig. 62). A mature mRNA containing 4 exons - 1,2,3,4 is formed from the primary transcript. Exons #5 and 6 were read as introns and excised. Based on this and RNA, calcitonin is synthesized. In brain cells, from the primary transcript containing 6 exons, a mature mRNA is formed, consisting of 5 exons - 1,2,3,5,6. The fourth exon was excised as an intron. Such mRNA controls the synthesis of a calcitonin-like protein responsible for taste perception.

Another geneIcarus(named after the legendary Icarus) is able to provide, through alternative splicing, the synthesis of 6 different polypeptides. In addition, polypeptides form between themselves in the cell about 20 different ensembles of the same polypeptides or different ones.

Violation of the splicing mechanism can lead to pathological conditions that are common name thalassemia. These include diseases associated with partial or complete suppression of the synthesis of one of the hemoglobin chains (α- or β-chains). For example, diseases associated with a lack of synthesis of the β-chain of hemoglobin can result from mutations in two regions of the gene encoding the β-chain - in the site responsible for polyadenylation and in one of the introns. In the first case, the process of formation of the polyadenyl tail is disrupted and an inferior β-chain of hemoglobin is formed. In the second case, the spliosome is unable to cut out the damaged intron, and the mature hemoglobin β-chain mRNA is not formed. In any case, the normal function of red blood cells will be significantly impaired.

MZ. Processing (or RNA maturation) is the process of converting newly synthesized, inactive RNA (pro-mRNA) into functionally active RNA. The process is associated with structural and chemical modifications of pro-mRNA. Occurs in the nucleus until the release of RNA into the cytoplasm. It consists of several stages: attachment of pro-mRNA to a protein, methylation of some bases, marking of one of the ends, polyadenylation of the other (opposite) end, excision of introns and stitching of exons. The last two processes are called splicing.

Questions for exams.

1. How do enzymes determine most of the places where there is damage to the DNA molecule?

ANSWER. In most cases, local denaturation occurs at the site of damage to the DNA molecule. It is determined by enzymes.

2. What happens at the site of damage to the DNA molecule?

ANSWER. Local denaturation occurs at the site of injury.

3. On the basis of what do repair enzymes restore the necessary sequence of nucleotides at the site of damage to one DNA strand?

ANSWER. Based on the principle of complementarity to the nucleotides of the opposite section of the DNA strand.

4. On the basis of what does DNA polymerase correctly fill gaps in a damaged DNA strand with nucleotides?

ANSWER. Based on the principle of complementarity of the nucleotides of the built-up chain to the nucleotides of the opposite strand.

5. What type of repair is carried out by an enzyme that is activated by a photon?

ANSWER. Photoreactivation.

6. What enzyme carries out repair using the energy of the sun?

ANSWER. Photolyase.

Which enzyme is directly involved in the synthesis of the RNA molecule?

ANSWER. DNA-dependent RNA polymerase or RNA polymerase.

List the periods of transcription.

ANSWER. Initiation, elongation, termination.

What are the components of the initiation complex during transcription?

ANSWER. From a special protein deposited on the promoter, RNA polymerase and transcription factors.

9. What is the name of the DNA region where the initiation complex is formed during transcription?

ANSWER. on the promoter.

10. What is the name of the nucleotide sequence in prokaryotes, which is determined by a special protein that precipitates on the promoter during the period of transcription initiation?

ANSWER. Block Pribnov.

11. What is the name of the nucleotide sequence in eukaryotes, which is determined by a special protein that precipitates on the promoter during the period of transcription initiation?

ANSWER. TATA box.

12. Where in the DNA molecule is the Pribnov block located in prokaryotes?

ANSWER. on the promoter.

13. Where in the DNA molecule is the TATA box located in eukaryotes?

ANSWER. on the promoter.

14. What is the name of the enzymatic complex that forms the transcriptional eye?

ANSWER. initiator complex.

15. What is the name of the section of the DNA molecule from which RNA synthesis begins?

ANSWER. Starting point, transcription start site.

16. Name the nucleotides that are in the terminator and possibly participate in the termination of transcription.

ANSWER. G, C.

17. Name the secondary structure in the terminator, which may be involved in the termination of transcription,

ANSWER. Hairpin.

18. What are the names of the codons located in the terminator and possibly involved in the termination of transcription.

ANSWER. Nonsense (nonsense) codons.

Immediately after synthesis, the primary RNA transcripts, for various reasons, do not yet have activity, are "immature" and subsequently undergo a series of changes, which are called processing. In eukaryotes, all types of pre-RNA are processed; in prokaryotes, only rRNA and tRNA precursors are processed.

Messenger RNA precursor processing

During transcription of DNA segments that carry information about proteins, heterogeneous nuclear RNAs are formed, which are much larger than mRNA. The fact is that, due to the mosaic structure of genes, these heterogeneous RNAs include informative ( exons) and non-informative ( introns) sections.

1. Splicing splice- glue butt) - a special process in which, with the participation small nuclear RNA introns are removed and exons are preserved.

Sequence of splicing events

2. Capping cap- cap) - occurs even during transcription. The process consists in attaching to the 5'-triphosphate the terminal nucleotide of the pre-mRNA 5'-carbon N 7 -methyl-guanosine.

The "cap" is necessary to protect the RNA molecule from exonucleases working from the 5' end, as well as to bind mRNA to the ribosome and to start translation.

3. Polyadenylation- with the help of polyadenylate polymerase using ATP molecules, from 100 to 200 adenyl nucleotides are attached to the 3 "end of RNA, forming a polyadenyl fragment - a poly (A) tail. The poly (A) tail is necessary to protect the RNA molecule from exonucleases, working from the 3" end.

Schematic representation of messenger RNA after processing

Ribosomal RNA precursor processing

rRNA precursors are larger molecules than mature rRNAs. Their maturation is reduced to the cutting of preribosomal RNA into smaller forms, which are already directly involved in the formation of the ribosome. There are four types of rRNA in eukaryotes 5S-, 5.8S-, 18S- and 28S-rRNA. At the same time, 5S-rRNA is synthesized separately, and large preribosomal 45S-RNA is cleaved by specific nucleases with the formation of 5.8S-rRNA, 18S-rRNA and 28S-rRNA.

In prokaryotes, ribosomal RNA molecules are completely different in their properties (5S-, 16S-, 23S-rRNA), which is the basis for the invention and use of a number of antibiotics in medicine.

Transfer RNA precursor processing

1. Modification of nucleotides in a molecule by deamination, methylation, reduction.
For example, the formation of pseudouridine and dihydrouridine.

The structure of modified uridyl nucleotides

2. The formation of an anticodon loop occurs by splicing

T ERMINATION

RNA polymerase will stop when it reaches the stop codons. With the help of a protein termination factor, the so-called ρ-factor (Greek ρ - "ro"), an enzyme and a synthesized RNA molecule are separated from the DNA matrix, which is primary transcript, a precursor of mRNA or tRNA or rRNA.

P ROCESSING RNA

PROCESSING OF MRNA PRECURSOR

And uninformative ( introns) sections.

1. Splicing (English splice - stick together end-to-end) is a special process in which, with the participation of small nuclear RNAs, introns are removed and exons are preserved.

2. Capping (English cap - cap) - occurs even during transcription. The process consists in attaching the terminal nucleotide of the pre-mRNA to the 5'-carbon of N7-methyl-guanosine to the 5'-triphosphate.

The "cap" is necessary to protect the RNA molecule from exonucleases working from the 5' end, as well as to bind mRNA to the ribosome and to start translation.

3. Polyadenylation- with the help of polyadenylate polymerase using ATP molecules, from 100 to 200 adenyl nucleotides are attached to the 3 "end of RNA, forming a poly (A) tail. The poly (A) tail is necessary to protect the RNA molecule from exonucleases working with 3 "-end.

P ROCESSING OF RRNA PRECURSOR

rRNA precursors are larger molecules than mature rRNAs. Their maturation is reduced to the cutting of preribosomal RNA into smaller forms, which are already directly involved in the formation of the ribosome. Eukaryotes have 5S-, 5.8S-, 18S-, and 28S-rRNA. At the same time, 5S-rRNA is synthesized separately, and large preribosomal 45S-RNA is cleaved by specific nucleases with the formation

5,8S-rRNA, 18S-rRNA, and 28S-rRNA.

At prokaryotic ribosomal RNA molecules are completely different in their properties(5S-, 16S-

23S-rRNA), which is the basis for the invention and use of a number of antibiotics in medicine

P ROCESSING THE PRECURSOR T RNA

1. Formation at the 3" end of the C-C-A sequence. For this, some pre-tRNA from the 3' end extra nucleotides are removed until the triplet is "exposed" C-C-A, others are joining this sequence.

2. Formation of an anticodon loop occurs by splicing and removal of an intron in the middle part of the pre-tRNA.

3. Nucleotide modification in the molecule by deamination, methylation, reduction. For example, the formation of pseudouridine and dihydrouridine.

In prokaryotic organisms, primary transcripts of α-coding genes begin to be used as templates for protein synthesis even before transcription is complete. Transcription and translation in prokaryotes is a coupled process. Transcripts of prokaryotic rRNA and tRNA are much longer than the corresponding mature RNA molecules. Thus, many tRNA transcripts contain more than one tRNA molecule. Thus, for prokaryotic organisms, the processing of primary rRNA and tRNA transcripts is necessary step formation of mature molecules.

Practically all primary RNA transcripts in eukaryotes undergo complex processing in the period between their synthesis and the beginning of their respective functions, either as mRNAs or as independent structural factors, such as or tRNAs. Processing occurs primarily in the core itself. Processing includes capping, cleavage and ligation reactions, addition of additional terminal nucleotides, and nucleoside modification. From 50 to 75% of mammalian nuclear RNA, including 5-capped strands, is not further included in the cytoplasmic mRNA. This amount of intranuclear loss of RNA is much higher than that which can be calculated considering only the removal of non-coding regions of the transcripts (see below). The exact function of the "excess" RNA in the nucleus of mammalian cells remains unknown.

Thanks to the development of methods of restriction mapping and sequencing of DNA molecules, it was possible to establish that in many eukaryotic genes between exons (i.e., coding fragments of the sequence) there are extended sections of DNA that do not carry genetic information directly translated into the amino acid sequence of proteins (see Chapter 38 ). Such intermediate sequences, or introns, are found in most, but not all, genes in higher eukaryotes. Primary transcripts of structural genes also include regions corresponding to nitrons. In a process called "splicing", these portions of the primary transcript are precisely excised and the corresponding exons are spliced together. The process takes place in the nucleus, then the formed mRNA molecules enter the cytoplasm, where they are translated (Fig. 39.9).

Until now, the exact mechanisms of error-free excision of introns and stitching of exons, as well as transport of RNA into the cytoplasm, are still unknown. However, research recent years gave a lot of new information about these processes. Although the nucleotide sequences in introns are very heterogeneous even within the same transcript, it is possible to identify a consensus sequence for each of the two intron-exon junctions (splicing sites) (Fig. 39.10). The consensus sequence of splicing sites at the intron-exon interface is not so

Rice. 39.9. Location of coding and non-coding sequences (introns) in the chicken ovalbumin gene structure. In the figure, the information segments that make up the mature mRNA are numbered and highlighted in black. The primary transcript begins to the left of the L-exon and ends in the 3-untranslated region behind exon 7. The structure of the mature mRNA is shown in the lower part of the figure; above it are the exon numbers, and below it are the serial numbers of nucleotides, the translation initiation point, and the position of the stop codon.

is unique in order to guarantee its strictly specific cleavage solely through the action of some specialized endonuclease. The small nuclear RNA present in significant amounts (U1 RNA) contains a sequence complementary to the splicing site consensus sequence (Fig. 39.11). In addition, U1-RNA molecules in the eukaryotic nucleus have been found to specifically bind to certain protein factors. Such RNA-protein complexes selectively associate with the 5- and 3-sequence splicing sites in the RNA. Antibodies against the U1 protein complex inhibit the excision of introns in vitro.

Interestingly, in patients with an autoimmune disease - systemic lupus erythematosus - antibodies were found against some specific U1-protein complexes. How this is directly related to the disease itself is not yet clear.

It has recently been found that during the removal of introns from mRNA precursor molecules, an unusual loop-like structure is formed. It turned out that the 5-end of the intron sequence is connected by a T-5-phosphodiester bond with adenylate located at a distance of 28-37 nucleotides from its 3-end. This process and the corresponding structures are shown schematically in Fig. 39.12.

As for the riddle of relationships and the corresponding mature ones, it can already be considered solved. Heterogeneous nuclear RNA consists of primary transcripts plus molecules located on early stages processing, which, after capping, adding a poly A tail, and removing introns, are transported into the cytoplasm already in the form of mature

Processing is another potential point of regulation of gene expression. Thus, the possibility of alternative splicing for the same primary transcript was demonstrated. For example, mRNA of a-amylase from salivary glands and from rat liver differ from each other in the structure of the 5-terminal sections of the sequence. The rest of the mRNA, including the coding region and the polyadenylation site, are identical. Further analysis showed that in order to attach two different capped leader sequences to the same mRNA "body"

Rice. 39.11. The proposed mechanism for identifying the splice site upon removal of introns from forms a complementary complex with the distal end of the consensus sequence of the splice site at the 3-end of exon a. The other end interacts with the splicing consensus site of exon b. The structure indicated by the line of dots is excised and the molecule is cross-linked along the G residues (shaded box).

Rice. 39.12. Putative Splicing Pathway Cleavage at the α-site is accompanied by loop formation and its subsequent release by cleavage from exon b. The intron is shown by a line, exons a and b by squares. These reactions occur with the participation of those united in a strong complex, which is part of the ribonucleoprotein structure, which is called the "spliceosome".

different splicing sites are used. Another example of alternative splicing is the formation of mRNA molecules encoding two immunoglobulin heavy chains. One mRNA molecule encodes a membrane-bound heavy chain, while the other encodes a secreted heavy chain (see Chapter 41). Thus, splicing is necessary for the formation of mature mRNA molecules and, in addition, can be used as one of the mechanisms of differential gene expression.

As it turned out, at least one of the forms of P-thalassemia, a disease in which the level of expression of one of the hemoglobin chains is markedly reduced, is the result of a nucleotide substitution at the intron-exon border, which prevents the removal of the intron and leads to a decrease or complete suppression of the synthesis of P- chains.

Messenger RNA (mRNA)

As mentioned above, mammalian molecules contain a capping structure at the 3' end and in most cases a polyadenylate tail at the 3' end. The capping structure is added to the mRNA in the nucleus prior to the transfer of the mRNA to the cytoplasm. The polyA structure is attached to the 3" end of the transcript either in the nucleus or in the cytoplasm. Secondary methylation of the mRNA molecule, including 2-hydroxyl groups and N6 atoms of adenylate

residues occurs after the transition of the RNA molecule into the cytoplasm. This process can also take place in the nucleus and play a certain role in splicing. The capping structure appears to be required for the formation of the nucleoprotein complex, which in turn is required for splicing. In addition, it may be involved in transport and translation initiation.

The function of the polyadenylate "tail" of mRNA is unknown. In many cases, the presence or absence of poly A is not directly related to transport into the cytoplasm, since not all polyadenylated heterogeneous nuclear RNAs enter the cytoplasm and not all cytoplasmic RNAs are polyadenylated. In mammalian cells, during the processes taking place in the cytoplasm, the polyadenylate “tails” of mRNA can both lengthen and shorten.

The turnover of poluA-containing mRNA in cultured mammalian cells is a first order process with a value close to the doubling time of the number of cells in culture. The degradation kinetics of histone-free poly A structures is a zero-order process characterized by an age-dependent decay with a lifetime of about 6 hours. It is still unclear whether these differences are associated with the presence or absence of terminal poluA sequences or with some other structural features of mRNA molecules of this class.

The size of cytoplasmic mRNA molecules, even after the removal of the poly A chain, remains significantly larger (often 2–3 times) than is required to encode the corresponding polypeptide. There are excess untranslated regions at both the 5- and 3-terminals of the translated region, with the 3-untranslated region generally reaching a greater length. The exact function of these sequences is unknown; there are reasons to believe that they are involved in the processing, transport, degradation, and translation of RNA.

Transfer RNAs (tRNAs)

Molecules as described in Chap. 37 and 40 act as adapter molecules when translated into protein sequences. The molecules contain many unusual ("minor") nucleic bases. Some of them are methylated derivatives of conventional bases, others contain unconventional glycosidic bonds. Both pro- and eukaryotic tRNA molecules are initially transcribed as large precursors, which often contain more than one tRNA molecule, subjected to nucleolytic processing by a special class of ribonucleases. In addition, the genes of some tRNAs contain a single intron 10-40 nucleotides long, located immediately before the site corresponding to the anticodon arm. Therefore, the processing of primary transcripts of many tRNA molecules must include the step of intron removal and precise splicing in the codon recognition region. This step is critical for tRNA to function as adapter molecules in protein synthesis. The nucleolytic processing of tRNA precursors seems to be directed not by the nucleotide sequence itself, but by a special three-dimensional structure that tRNA molecules can form, and therefore is realized only for molecules capable of folding into certain functional structures.

Further modifications of tRNA molecules include nucleotide alkylation and addition of a characteristic CCA triplet to the 3-terminus of the molecule. This triplet serves as the point of attachment of the corresponding amino acid directed by this tRNA molecule to the polypeptide synthesis reaction. Methylation of mammalian tRNA precursors probably occurs in the nucleus, while cleavage and addition of the CCA triplet occurs in the cytoplasm, since the turnover rate for the terminal part of the tRNA is higher than for the molecule as a whole. Attachment of an amino acid to the CCA terminus requires certain enzymes in the cytoplasm of mammalian cells.

Ribosomal RNA (rRNA)

In mammalian cells, ribosomal RNA molecules (two major and one minor) are transcribed as part of a large common primary transcript (Fig. 39.13). The processing of this transcript with the formation of mature ones transported to the cytoplasm occurs in the nucleolus, where the ribosomal RNA genes themselves are localized. There are hundreds of copies of these genes in every cell. Transcriptional units contain sequences located one after another in the 5-3 direction. The primary transcript is extensively methylated directly in the nucleolus. In this α-precursor, the corresponding region contains 65 methylated ribose residues and 5 methylated nucleobases. Methylation occurs only at sites that later form mature rRNA molecules. The 458 precursor undergoes nucleolytic processing, however, the processing signals are markedly different from the corresponding signals in It is likely that the processing mechanism also differs from the nucleolytic processing mechanism during maturation.

Rice. 39.13. Scheme of the formation of mature ribosomal RNA during the processing of RNA precursor molecules. Final product marked with black rectangles. (Reproduced, with permission from Perry R. P.: Processing of RNA Annu. Rev. Biochem. 1976, 45:605.)

Almost half of the original primary transcript (Fig. 39.13) is degraded. During processing in the nucleolus, further methylation occurs, and in the same place, by binding to ribosomal proteins, it forms a large α-subunit of the ribosome. The molecule is also formed in the nucleolus and is an integral part of the large ribosomal subunit. The molecule in combination with a set of corresponding polypeptides forms a small α-subunit of the ribosome.