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Definition of nucleotide-associated proteins and RNA-polymerase associated proteins

Definition of nucleotide-associated proteins and RNA-polymerase associated proteins



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Nucleotide-associated proteinsandRNA-polymerase associated proteins, I found those two terms in an article but I didn't understand what they mean exactly, is there any relation between those terms andDNA-binding proteins?

The article : DNA supercoiling-a global transcriptional regulator for enterobacterial growth?


In short:

Nucleotide-associated proteins are DNA-binding proteins that bind DNA. RNA-polymerase associated proteins bind RNA-polymerases and are required for its functionality.

In more detail:

DNA-binding protein is a higher level term that comprises all proteins that bind to DNA. These can be seen as nucleotide-associated proteins as they interact with the nucleotides in the DNA strand, even though I did not hear people use that term very often. There are two types of binding:

  • Non-specific DNA-binding proteins that do not bind to specific sequences DNA sequences, e.g. structural proteins.

  • Specific DNA-binding proteins that bind to specific DNA sequences, e.g. transcription factors. These proteins mostly have specific DNA binding motifs like zinc fingers or helix-turn-helix motifs.

RNA-polymerase (RNAP) associated proteins are different from that. RNAP itself is a DNA-binding protein, but proteins binding to RNAP are necessary for it to be fully functional. The fully functional RNAP then is a holoenzyme. For example RNAP II requires an initiation factor called sigma in bacteria and several general transcription factors and regulatory proteins in eukaryots.


Neither of the terms “Nucleotide-associated protein” or “RNA-polymerase associated protein” are standard in molecular biology in so far as they are not defined in the reference Gene Ontology. (In contrast, “DNA-binding” is.)

An internet search does not bring up many examples of the former usage (at least) and in those it brings up it is not defined. One can only conclude that it means exactly what it says and no more, presumably because the author has limited information about the function of the protein.(Without the original reference I cannot be sure.)

A “Nucleotide-associated protein” is therefore “a protein that in some way interacts with a nucleotide”. On may imagine that it might bind the nucleotide, but the nature of this binding is unspecified and could be reflect the fact that the protein is a kinase of some sort and ATP (or GTP etc) is a substrate. It therefore need not be a DNA-binding protein, in contrast to the statement in the accepted answer from @AlexDeLarge. In fact the term would most likely imply a mononucleotide-binding protein, i.e. a protein that bound ATP or cyclicAMP or GDP or dUDP etc. If it bound a polynucleotide, this could equally well be RNA as DNA.

Likewise, an “RNA-polymerase associated protein” is “a protein that in some way interacts with RNA-polymerase”. It is more likely that this is a protein that forms a complex with - i.e. binds to - RNA-polymerase, but the fact that the author does not use the term “binding” suggests that he is being cautious because he has no evidence for this. Perhaps both proteins are found in the same immunoprecipitate.


Describe what is happening during transcription elongation and termination

  • RNA polymerase II (RNAPII) transcribes the major share of eukaryotic genes.
  • During elongation, the transcription machinery needs to move histones out of the way every time it encounters a nucleosome.
  • Transcription elongation occurs in a bubble of unwound DNA, where the RNA Polymerase uses one strand of DNA as a template to catalyze the synthesis of a new RNA strand in the 5&prime to 3&prime direction.
  • RNA Polymerase I and RNA Polymerase III terminate transcription in response to specific termination sequences in either the DNA being transcribed (RNA Polymerase I) or in the newly-synthesized RNA (RNA Polymerase III).
  • RNA Polymerase II terminates transcription at random locations past the end of the gene being transcribed. The newly-synthesized RNA is cleaved at a sequence-specified location and released before transcription terminates.

Viral polymerases

Viral polymerases play a central role in viral genome replication and transcription. Based on the genome type and the specific needs of particular virus, RNA-dependent RNA polymerase, RNA-dependent DNA polymerase, DNA-dependent RNA polymerase, and DNA-dependent RNA polymerases are found in various viruses. Viral polymerases are generally active as a single protein capable of carrying out multiple functions related to viral genome synthesis. Specifically, viral polymerases use variety of mechanisms to recognize initial binding sites, ensure processive elongation, terminate replication at the end of the genome, and also coordinate the chemical steps of nucleic acid synthesis with other enzymatic activities. This review focuses on different viral genome replication and transcription strategies, and the polymerase interactions with various viral proteins that are necessary to complete genome synthesis.

Figures

Baltimore virus classification scheme based…

Baltimore virus classification scheme based on genome type and method of mRNA synthesis…

Two-metal mechanism used by polymerases…

Two-metal mechanism used by polymerases to catalyze the nucleotidyl transfer reaction. Template, primer,…

Polymerase sequence motifs in RNA-dependent…

Polymerase sequence motifs in RNA-dependent and DNA-dependent polymerases. ( a ) Alignment of…

Schematic diagram of initiation mechanisms…

Schematic diagram of initiation mechanisms of viral polymerases. ( a ) Primer-dependent initiation.…

Poliovirus genome replication. ( a…

Poliovirus genome replication. ( a ) Genome replication steps. (i) VPg uridylation. Terminal…

Crystal structures of 3D polymerase…

Crystal structures of 3D polymerase that use VPg-linked primers. ( a ) FMDV…

Flavivirus genome replication. ( a…

Flavivirus genome replication. ( a ) Minus-strand RNA synthesis. The viral RdRp uses…

The Flaviviridae RNA-dependent RNA polymerases…

The Flaviviridae RNA-dependent RNA polymerases that use a de novo initiation mechanism. (…

Domain arrangement of RNA-dependent RNA…

Domain arrangement of RNA-dependent RNA polymerases from negative-sense ssRNA viruses. Influenza virus polymerase…

Reverse transcription of the HIV-1…

Reverse transcription of the HIV-1 genome. ( a ) Minus-strand DNA synthesis. Minus-sense…

Structures of HIV reverse transcriptase.…

Structures of HIV reverse transcriptase. ( a ) p66 subunit ( top )…

ϕ29 protein-primed DNA replication. The…

ϕ29 protein-primed DNA replication. The linear dsDNA genome of ϕ29 has a terminal…

Structures of ϕ29 DNA-dependent DNA…

Structures of ϕ29 DNA-dependent DNA polymerase. ( a ) ϕ29 polymerase. The N-terminal…

RNA transcription by T7 DNA-dependent…

RNA transcription by T7 DNA-dependent RNA polymerase. ( a ) Initiation of RNA…

T7 RNA polymerase initiation and…

T7 RNA polymerase initiation and elongation complexes. ( a ) Initiation complex containing…

Schematic of T7 polymerase reaction…

Schematic of T7 polymerase reaction cycle ( top ) and structures of T7…


Why is RNA important?

DNA and RNA are ancient, but their discovery was relatively recent. In 1869, chemist Friedrich Miescher documented a kind of molecule that had never been studied before—nucleic acid. It wasn’t until around the 1930s that the term DNA began to be used, with RNA following in the 1940s. In the 1950s, the work of biophysicist Rosalind Franklin and biologists James Watson and Francis Crick revealed DNA’s double helix structure. The function of RNA began to be further understood during the 1950s and 60s as scientists began to understand the role of messenger RNA.

This understanding is still developing. Until quite recently, RNA’s role was thought to be largely limited to assisting with protein synthesis in its forms as messenger RNA, transfer RNA, and ribosomal RNA. However, scientists are continuing to discover new types of RNA and more functions that RNA performs in the body. For example, recent discoveries suggest that there are several types of RNA that regulate how many proteins the ribosomes produce.


Shared Flashcard Set

Which of the following is untrue of DNA?

D. It remains constant among different species

E. It has variability between species

D. It remains constant among different species

Which of the following is not a nucleotide found in DNA?

If you were a biologist and you were studying purines, which of the following molecule(s) would you be studying?

Which of the following would not be found in DNA?

Which of the following best describes the chemical composition of a nucleotide?

A. a nitrogen-containing base and a pentose sugar

B. a nitrogen-containing base, a phosphate and a pentose sugar

C. a phosphate and a pentose sugar

D. a nitrogen-containing base, a phosphate, and a hexose sugar

B. a nitrogen-containing base, a phosphate and a pentose sugar

A biologist is studying the process of how DNA is copied. Which of the following would he/she be studying?

During the process of transcription, the information in:

A. Protein is converted to RNA information

B. RNA is converted into protein information

C. RNA is converted into DNA information

D. DNA is converted into RNA information

E. DNA is converted into protein information

D. DNA is converted into RNA information

Which of the following processes does not take place during translation?

A. Attachment of a ribosome to mRNA

B. The number of codons in mRNA

C. The enzyme that attaches the amino acid to tRNA

D. The proteins associated with rRNA

E. The sequence of anticodons

E. The sequence of anticodons

Which is most directly responsible for the sequence of amino acids in a protein?

A. the sequence of codons in mRNA

B. t he number o f codons in mRNA

C. the enzyme that attaches the amino acid to tRNA

D. the proteins associated with rRNA

E. the sequence of anticodons

A. the sequence of codons in mRNA

Each codon is made up of this number of bases:

A biochemist is studying the process of how a protein chain is assembled. Which of the following would they be studying?

If you were trying to isolate the molecule which carries genetic information from the nucleus to where proteins are manufactured, you would be studying whcih of the following molecules?

The function of transfer RNA is to:

A. carry amino acids to ribosomes

B. transfer nucleotides to the nucleus

C. transmit coded information to the cytoplasm

E. act as the site for protein synthesis

A. carry amino acids to ribosomes

The ________ of a tRNA molecule will attract the codon of an mRNA colecule.

C. a mino ac id binding site

In modern biochemical genetics, the flow of inherited information is from:

Which of the following cannot be an enviromental mutagen?

E. all are considered mutagens

Which series is arranged in order from largest to smallest in size?

A. chromosome, nucleus, cell, DNA, nucleotide

B. cell, nucleus, chromosome, DNA, nucleotide

C. nucleotide, chromosome, cell, DNA, nucleus

D. cell, nucleotide, nucleus, DNA, chromosome

E. DNA, nucleotide, nucleus, cell, chromosome

B. cell, nucleus, chromosome, DNA, nucleotide

Nitrogen bases are held together by weak bonds called:

What type of bonds bind the sugar and phosphate molecules?

Amino acids are bond together to form proteins by what type of bonds?

Match the following types of chromosomal mutations with the right sequence change.

Why is the genetic code called a "unviersal code"?

because it works with everything, every living thing contains it, and the same nitrogen bases are in all living things.


Quorum Sensing

Marijke Frederix , J. Allan Downie , in Advances in Microbial Physiology , 2011

7.1.3 Nitrogen Limitation

Sigma factors are subunits of RNA polymerase required for gene transcription to occur. The expression of most genes in a bacterial cell is dependent on the expression of the ‘housekeeping’ sigma factor σ 70 , but bacteria can express different sigma factors in response to different environmental conditions. These alternative sigma factors are involved in adaptation to specified niches, such as interactions with eukaryotic hosts. In many bacteria a link between one of these alternative sigma factors and QS gene regulation has been found. Under nitrogen starvation conditions the alternative sigma factor RpoN (σ 54 ) is activated and induces the expression of genes that are involved in nitrogen assimilation ( Hendrickson et al., 2001 ).

In V. cholerae and V. harveyi, the activity of the response regulator LuxO-P requires RpoN to induce the transcription of the Qrr sRNA's ( Klose et al., 1998 Lilley and Bassler, 2000 Lenz et al., 2004 ). Increased transcription of the qrr genes causes a destabilization of the QS master regulator, and thus RpoN has a negative effect on the expression of QS-regulated genes ( Fig. 5 A and B).

RpoN was reported to reduce production of RhlI and LasI-made AHLs in P. aeruginosa probably due to indirect effects, as RpoN induced expression of vfr and repressed expression of gacA ( Heurlier et al., 2003 ). In contrast RpoN was observed to increase production of RhlI-made AHLs by induction of rhlI expression ( Thompson et al., 2003 ) and RpoN activated expression of rhlR ( Medina et al., 2003b ).


Definition of Nuclear Pore

The nuclear pores can define as the tiny orifices that help the nucleus to communicate with the cytoplasm by directing the nucleocytoplasmic exchange for cell coordination. The small ions and molecules (smaller than 5000 Daltons) can diffuse freely within the cell, while specific biomolecules can travel up and down the cell via protein-mediated or active transport. Nuclear pores have a diameter and thickness of 120nm and 50nm, respectively.

Structure of Nuclear Pore Complex

The cylindrical multiprotein complexes that surround each nuclear pore and direct the nucleocytoplasmic exchange are termed as NPCs. Both the concentric membranes of the nuclear envelop fuses with the multiprotein complex that is manifested by 30 disparate proteins termed as NUPs or Nucleoporins. The nuclear pore complex possesses an octagonal symmetry. The structure of the nuclear pore complex comprises the following key elements:

Nucleoporins scaffold: The nucleoporins mainly constitute the formation of the nuclear pore complex that is massively entangled to enable selective exchange within the cell. It comprises the cytoplasmic and nucleoplasmic ring, in between which a central spoke ring is organized. NPC appears as an octagonal ring due to scaffolding of few nucleoporins.

Central channel or transporter: Some nucleoporins like FG repeats account for the formation of central channel. The nucleoporins of central channel functions as a selective barrier, which only allows import and export of large biomolecules across the bilayer nuclear envelop that carries specific amino acid sequence. It is 36-38 nm wide. The central channel is encased by the eightfold symmetrical spokes.

Cytoplasmic filaments: The short and thick stringy structures are associated with the cytoplasmic ring are called cytoplasmic filaments. It has a diameter of 3.3 nm and extended towards the cytoplasm. It functions like a sensor that specifically binds with the signal proteins attached to the molecules that have to be imported into the nucleus. These are eight in number and covers less space towards the cytoplasmic end.

Nuclear basket: A large bin-like structure is associated with the nuclear ring. It enables tethering of nucleoporins inwards the nucleus lumen. This basket plays a significant role in the export of biomolecules.

Transport of Macromolecules

A nucleus is surrounded by a selective bilayer nuclear membrane that acts as a barrier for the macromolecules of size <50 kDa but permits free diffusion of small ions or metabolites. But, macromolecules like RNA polymerase, histone proteins can enter into the nucleus, and the biomolecules like RNA and ribosomal subunits can be exported into the cytosol through active transport.

Thus, the exchange between the nucleus and cytoplasm is bidirectional that allows to and fro movement of nucleoplasmic and cytoplasmic contents. One thing we should always remember that the necessary macromolecules can only move in and exit the nucleus, which contains a discrete amino acid sequence.

The molecules possessing specific amino acid sequence are tagged either by the nuclear localization (NLS) or nuclear export signals (NES). For selective transport, specific nuclear receptors also binds with the specific sequence of amino acid on the cargo proteins and stabilize the complex. RAN is another group of proteins that provides free energy for the active transport of macromolecules.

RNA polymerase and histone proteins are tagged with nuclear localization signals that act as a binding site for an importin (nuclear importin receptor), which in turn import these macromolecules into the nucleus. Therefore, nuclear localization signals direct importin proteins to the nucleus.

The NLS was first observed in the viral protein (T-antigen) that is needed for the viral replication inside the nucleus of the host cell. Nuclear localization signals are the non-cleavable amino acid sequences that are rich in lys-residues.

RNA and ribosome subunits are tagged with nuclear export signals that act as a binding site for an exportin (nuclear exportin receptor), which in turn export these macromolecules into the cytoplasm. Thus, nuclear export signals direct exportin proteins to the cytosol. These are the short amino acid sequences that are rich in leu-residues.

Nuclear Receptor Proteins

Particular nuclear signals will interact with specific receptor proteins. NLS will mainly interact with the nuclear exportin receptor, while NES interacts with the nuclear importin receptor. Exportins associate with the macromolecules inside the nucleus and release them into the cytosol. Oppositely, importins interact with the molecules in the cytosol and release them into the nucleus.

Both importin and exportin are the nuclear receptor proteins that are highly homologous in sequence and included in the family of karyopherins. At the time of translocation, the importins and exportins combine with the FG region on nucleoporins. A region that comprises phe-gly repeats is called FG-repeats.

RAN Protein

Apart from nuclear receptor proteins, there is another protein named RAN that contains small monomeric nuclear GTPases. RAN protein generally exists in two conformations, namely RAN-GTP complex and RAN-GDP complex. The occurrence of RAN-GTP and RAN-GDP primarily exists within the nuclear lumen and the cytosol, respectively.

The above two complexes differ due to an asymmetric distribution of two proteins, namely GEF and GAP. GEF or guanine nucleotide exchange factor resides in the nucleus that replaces the Ran-GDP into Ran-GTP. Oppositely, the GTPase activating protein or GAP that exists within the cytosol causes hydrolysis of GTP into GDP, thus transforming the Ran-GTP to Ran-GDP.

The RAN-GAP protein confines atop of NPC, i.e. towards the cytoplasmic surface. The RAN-GDP and RAN-GTP stabilize the import and export complexes. Therefore, the RAN gradient fulfils the need of free energy for the active transport of macromolecules and also mediates directionality during the translocation.

Import and Export Cycle

The steps involved in the import and export cycle through the nuclear pore complex includes:

Nuclear Import Cycle

The nuclear importin receptor possesses α and β subunit. A protein cargo that is destined to be ejected inside the nucleus binds to the α-subunit through nuclear localization signal. The RAN-GDP complex that exists in the cytosol joins to the β-subunit of an importin receptor.

The above complex then binds to the nucleoporins of the NPC and finally shuttles beyond the lipid bilayer nuclear envelop. A protein cargo is then ejected into the nuclear lumen, and the remaining complex (RAN-GDP and importin) goes through conformational change within the nucleus.

The RAN-GDP interacts with the RAN-GEF, i.e. guanine nucleotide exchange factor that adds one phosphate group to the RAN-GDP complex to form RAN-GTP. An importin receptor is reclaimed back across the pore to the cytosol along with the RAN-GTP.

As we discussed earlier, the RAN-GAP (GTPase activating protein) are the proteins localized on the cytoplasmic side of NPC that cause hydrolysis of GTP. The GTPase enzyme causes deletion of one phosphate group from the RAN-GTP complex and results into dissociation of importin receptor and formation of RAN-GDP complex. Finally, an importin receptor becomes free to start another cycle.

Nuclear Export Cycle

The nuclear exportin receptor attaches with a target protein that is destined to be ejected into the cytosol. Then, the RAN-GTP complex that exists within the nucleus combines with the exportin receptor. The whole stable complex binds to the nucleoporins of the NPC and finally shuttles beyond the bilayer phospholipid nuclear envelop.

The RAN-GAP on the cytoplasmic surface of NPC causes a conformational change in the RAN-GTP by removing one phosphate group. As a result, a protein cargo dissociates from the complex in the cytosol. The remaining complex, i.e. RAN-GDP plus exportin receptor, is reclaimed back across the pore inside the nucleus.

The RAN-GEF is a protein domain found within the nucleus, which adds one additional phosphate group to the RAN-GDP complex. It results in the release of exportin receptor and formation of RAN-GTP complex. Finally, an exportin receptor becomes free to start a new cycle.

Conclusion

Let us suppose the nuclear pore complex as a barricade, the large biomolecules as people who must have their pass to enter and exit out of it. Similarly, the large molecules can only enter and exit by the means of nuclear pore complexes, which are tagged with nuclear receptor proteins.


SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum

Positive-strand RNA viruses, a large group including human pathogens such as SARS-coronavirus (SARS-CoV), replicate in the cytoplasm of infected host cells. Their replication complexes are commonly associated with modified host cell membranes. Membrane structures supporting viral RNA synthesis range from distinct spherular membrane invaginations to more elaborate webs of packed membranes and vesicles. Generally, their ultrastructure, morphogenesis, and exact role in viral replication remain to be defined. Poorly characterized double-membrane vesicles (DMVs) were previously implicated in SARS-CoV RNA synthesis. We have now applied electron tomography of cryofixed infected cells for the three-dimensional imaging of coronavirus-induced membrane alterations at high resolution. Our analysis defines a unique reticulovesicular network of modified endoplasmic reticulum that integrates convoluted membranes, numerous interconnected DMVs (diameter 200-300 nm), and "vesicle packets" apparently arising from DMV merger. The convoluted membranes were most abundantly immunolabeled for viral replicase subunits. However, double-stranded RNA, presumably revealing the site of viral RNA synthesis, mainly localized to the DMV interior. Since we could not discern a connection between DMV interior and cytosol, our analysis raises several questions about the mechanism of DMV formation and the actual site of SARS-CoV RNA synthesis. Our data document the extensive virus-induced reorganization of host cell membranes into a network that is used to organize viral replication and possibly hide replicating RNA from antiviral defense mechanisms. Together with biochemical studies of the viral enzyme complex, our ultrastructural description of this "replication network" will aid to further dissect the early stages of the coronavirus life cycle and its virus-host interactions.

Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1. The Coronavirus Replicase Polyprotein

Figure 1. The Coronavirus Replicase Polyprotein

The domain organization and proteolytic processing map of the…

Figure 2. Overview of Membrane Structures Induced…

Figure 2. Overview of Membrane Structures Induced by SARS-CoV Infection

Electron micrographs of SARS-CoV–infected Vero…

Figure 3. Electron Tomography Revealing the Interconnected…

Figure 3. Electron Tomography Revealing the Interconnected Nature of SARS-CoV–Induced DMVs

Figure 4. Electron Tomography of SARS-CoV–Induced CM,…

Figure 4. Electron Tomography of SARS-CoV–Induced CM, DMVs, and VPs

Figure 5. Electron Tomography of the SARS-CoV–Induced…

Figure 5. Electron Tomography of the SARS-CoV–Induced Reticulovesicular Membrane Network at a More Advanced Stage…

Figure 6. Immunogold EM of the SARS-CoV…

Figure 6. Immunogold EM of the SARS-CoV Replicase in Infected Cells

SARS-CoV–infected Vero E6 cells…

Figure 7. Detection of dsRNA in SARS-CoV–Infected…

Figure 7. Detection of dsRNA in SARS-CoV–Infected Cells

SARS-CoV–infected Vero E6 cells were fixed at…

Figure 8. Immunogold EM Reveals Abundant dsRNA…

Figure 8. Immunogold EM Reveals Abundant dsRNA Labeling on the Interior of SARS-CoV–Induced DMVs

Figure 9. Electron Tomography-Based Model of the…

Figure 9. Electron Tomography-Based Model of the Network of Modified ER Membranes That Supports SARS-CoV…


Medical Definition of RNA

&mdash called also ribonucleic acid

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Molecular Biology of Wound Healing: The Conditioning Phenomenon

1 The Activation of Genes after Wounding

Gene activation may mean that genes already active in intact tissues are copied at a much faster rate after wounding, or it may signify that genes that were blocked before injury are read out after injury. There is evidence that both may be true for wounded plant model systems. First of all, a tremendous increase in RNA content accompanies wound healing ( Kahl, 1973 , 1974a ). So there is no doubt, that synthesis really occurs. However, in most systems studied so far, there are two phases of RNA synthesis to be distinguished from one another. During the first 2–3 hr after wounding the synthesis of ribosomal RNA (rRNA) and transfer RNA (tRNA) is negligibly small both in Jerusalem artichoke and potato tuber tissues. In contrast, there is heavy incorporation of 3 H- and 14 C-labeled nucleotides into what is presumed to be messenger-RNA (mRNA). Actually labeling of the heterodisperse RNA starts immediately after injury in potato tuber ( Sato et al., 1978 ) and Jerusalem artichoke cells ( Byrne and Setterfield, 1977 ). Thus there seems to be a preferential transcription of unique sequences into mRNA. It should be stressed at this point that even resting cells do contain some mRNA. So do both Jerusalem artichoke and, to a lesser degree, potato tuber tissues. This pre-existing mRNA is quite obviously used for translation ( Byrne and Setterfield, 1978 Sato et al., 1980 ), but principally codes for the same proteins as that synthesized after wounding ( Sato et al., 1980 ). Therefore it seems as if the pattern of activated genes in the intact cells is retained shortly after wounding.

The newly synthesized mRNA becomes rapidly associated with ribosomes and certainly is responsible for both polysome formation as well as increased protein synthetic capacity ( Table I ). Both polyadenylated [poly(A) + ] and nonadenylated RNA's [poly(A) − ] are present on polysomes of wounded potato cells (R. K. Tripathi, personal communication Sato et al., 1980 ). They are able to program a wide variety of polypeptides in cell-free translation systems, such as derived from rabbit reticulocytes (R. K. Tripathi, personal communication) and wheat germs ( Sato et al., 1980 ). However, there are coding differences of the poly(A) + - and poly(A) − –RNA's. Some five distinct polypeptides (i.e., a 22,000 and 23,000 daltons component) are only encoded in the poly(A) + -RNA ( Sato et al., 1980 ). About 6 hr after wounding potato tuber tissues, the poly(A) + -RNA of polysomes in fact contributes about 70% to the translational capacity of total polysomal RNA. In spite of these qualitative differences, the molecular size distribution between the two RNA classes is quite similar, it ranges from about 6S to over 30S ( Sato et al., 1980 ). Messenger-RNA synthesis keeps on going for about 10–15 hr after wounding, which does not necessarily exclude the formation of special messages afterward (i.e., the messenger for suberin induction, see Section III , D , 3).

Starting at about 3–6 hr after wounding, label is also increasingly introduced into ribosomal RNA (rRNA). In wounded carrot root as well as Jerusalem artichoke tuber tissues, representative for all other plant tissues, a precursor of about 2.3 ×10 6 daltons is transcribed first, but is rapidly cleaved to yield a 1.4 ×10 6 and a 0.9 × 10 6 daltons component. These intermediate molecules are subsequently processed to RNA molecules of 1.3 ×10 6 and 0.7 × 10 6 daltons, respectively, the RNA's of the large and the small ribosomal subunits ( Rogers et al., 1970 Leaver and Key, 1970 ). The smaller 18S rRNA after its export from the nucleolus enters the cytoplasm more rapidly than the 25S rRNA, which leads to a higher specific activity of the smaller component in labelling experiments ( Kahl, 1971a ).

All these events are in good agreement with electron microscopic as well as autoradiographic data. Thus, nucleoli as the loci of ribosomal RNA transcription and processing should indicate the dramatic changes in rRNA synthesis after wounding. They do. In quiescent cells these organelles are relatively small and compact structures. Only a thin peripheral granular zone may be present, embedded in masses of specialized chromatin ( Rose et al., 1972 Jordan and Chapman, 1973 Barckhausen, 1978 ). Nucleolar vacuoles are small in number and size, if ever present ( Barckhausen, 1978 ). All features strongly point to an inactive nucleolus in intact tissues, a view which is definitely supported by biochemical analyses.

Soon after wounding, evident in about 2–4 hr, the nucleolar volume measurably increases up to a maximum at about 24 hr. Thereafter it declines ( Rose et al., 1972 Jordan and Chapman, 1971 , 1973 Kahl and Wielgat, 1976 Barckhausen, 1978 ). Distinct changes in structural organization accompany this increase in volume. A defined granular zone develops, which intermingles with the fibrillar regions. Nucleolar vacuoles appear or progressively increase in size and exhibit rhythmic pulsations over the whole wound-healing period. They may occur as “giant vacuoles” ( Barckhausen, 1978 ), which have been assigned the function of ribonucleoprotein transport into the cytoplasm. At the same time the nucleolar-associated chromatin (L-zone: Jordan and Chapman, 1971 ) greatly disperses, which is indicative for the active transcription of the ribosomal cistrons on this particular chromatin ( Setterfield et al., 1978 ). All the evidence, then, points to highly active nucleoli in wounded tissues.

Again, autoradiographic experiments revealed, that most label of administered [ 3 H]uridine is in the chromatin region, not in the nucleolus of freshly wounded Jerusalem artichoke cells. After only 1 hr, label appears also in the nucleolus. Quite clearly, the onset of rRNA synthesis after wounding is quite rapid. The processing of the precursor molecules, however, evidently is so time-consuming, that they do not appear as ribosomal constituents before 3–4 hr after injury ( Byrne and Setterfield, 1977 ).

Transfer-RNA (tRNA) synthesis is also a characteristic feature of wounded tissues ( Kahl, 1971a Frazer, 1975 ). Obviously the tRNA in intact tissues of sugar beet roots is undermethylated and hence probably inactive. Wounding induces a rapid and extensive methylation of the tRNA ( Stone and Cherry, 1972 King and Chapman, 1973 ), and at the same time the tRNA's develop an increased amino acid acceptor activity. So the rapid wound-induced methylation of pre-existing tRNA's may well be a factor in early protein synthesis.

Wounding then in some way must trigger the synthesis of various messenger, ribosomal, and transfer RNA's or, in other words, the readout of unique sequences as well as the multiple tDNA and rDNA genes. It is still an enigma how this is achieved both in animal and plant organisms. So all we know is that intact organs, in general, do not possess much of the capability to support DNA-dependent RNA synthesis, which is consequently low in intact wound model systems, such as sugar beet ( Duda and Cherry, 1971 ), Jerusalem artichoke ( Kamisaka and Masuda, 1971 ), and potato tuber ( Kahl and Wielgat, 1976 ). However, template availability for exogenous E. coli RNA polymerase by far exceeds the actual chromatin activity with endogenous polymerase. This may mean that endogenous RNA polymerase concentration is limiting, although this is far from conclusive. In any event, wounding activated DNA-dependent RNA polymerases in all model systems ( Duda and Cherry, 1971 Kahl and Wielgat, 1976 Kahl and Wechselberger, 1977 ), both RNA polymerase I (rRNA synthesis) and polymerase II (hnRNA and hence presumably mRNA synthesis) are affected. Polymerase I usually shows a vigorous, about fourfold increase in activity some 18 hr after wounding potato tuber tissue. Polymerase II only doubles its activity in the same period ( Kahl and Wielgat, 1976 ). It can be proved that the polymerases of wounded tissues read out a set of other genes than their counterparts in intact organs. This has conclusively been demonstrated for potato tuber ( Kahl and Wielgat, 1976 Kahl and Wechselberger, 1977 ) and sugar beet tissues ( Duda and Cherry, 1971 ) by nearest neighbor frequency analyses. Consequently, the overall base composition of the RNA synthesized on isolated chromatin in vitro is distinctly different between quiescent and wounded tissues. For example, the frequency with which AMP serves as nearest neighbor for AMP is definitely higher in wounded tissues: indicative of the wound-induced formation of poly(A) + -RNA ( Fig. 6 ).

Fig. 6 . Wound-induced changes in chromatin-bound protein phosphokinases (○—○) and DNA-dependent RNA polymerase I (open bars) and II (black bars) activity in potato tuber tissues. For nearest neighbor frequency analysis the RNA synthesized on purified chromatin in the presence of adenosine 5′-α-[ 32 P]triphosphate was isolated and hydrolized and the different 2′,3′-mononucleotides separated by high voltage paper electrophoresis. The distribution of label in each 2′,3′-mononucleotide (mole%) is presented below for resting (left) and wounded tissue after a 36-hr wound-healing period (right). Standard deviation ± 1.0 mole%.


Watch the video: A movie of RNA Polymerase II transcription (August 2022).