Rna polymerase synthesizes the rna transcript during the stage of transcription.

RNA and Protein Synthesis

All eukaryotic cells use DNA to direct protein synthesis. Proteins are made in the cytoplasm on the ribosome. These polypeptide-making factories contain more than 50 different proteins, as well as RNA. RNA is similar to DNA, and its presence in ribosomes suggests its important role in protein synthesis (Fig. 10.2). RNA differs from DNA in two ways: RNA contains ribose as sugar rather than the deoxyribose in DNA, and RNA contains the pyrimidine uracil (U in codon designations) instead of thymine.2 In addition, RNA does not have a regular helical structure. The class of RNA present in ribosomes is called ribosomal RNA (rRNA).3 rRNA and ribosomal proteins provide sites at which polypeptides are assembled. Transfer RNA (tRNA) transports the amino acids to the ribosome for the synthesis of polypeptide.4,5 There are more than 40 different tRNA molecules in human cells. tRNA is smaller than rRNA and is present in free form in the cytoplasm. Messenger RNA (mRNA) consists of long strands of RNA molecules that are copied from DNA. mRNA travels to the ribosome to direct the assembly of polypeptides.

RNA is synthesized on a DNA template by a process of DNA transcription in which RNA polymerase enzymes make an RNA copy of a DNA sequence. RNA polymerases are formed from multiple polypeptide chains with a molecular weight of 500,000.6,7 In eukaryotic cells there are three different types of RNA polymerases. RNA polymerase II transcribes the gene whose RNAs will be translated into proteins. RNA polymerase I makes the large rRNA precursor (45S rRNA) containing the major rRNAs. RNA polymerase III makes very small, stable RNAs, including tRNA and the small 5S rRNA. In mammalian cells there are approximately 20,000 to 40,000 molecules of each of the RNA polymerases.

Abstract

Manipulating RNA synthesis rates is a primary method the cell uses to adjust its physiological state. Therefore to design synthetic genetic networks and circuits, precise control of RNA synthesis rates is of the utmost importance. Often, however, a native promoter does not exist that has the precise characteristics required for a given application. Here, we describe two methods to change the rates and regulation of RNA synthesis in cells to create RNA synthesis of a desired specification. First, error-prone PCR is discussed for diversifying the properties of native promoters, that is, changing the rate of synthesis in constitutive promoters and the induction properties for an inducible promoter. Specifically, we describe techniques for generating diversified promoter libraries of the constitutive promoters PLtetO-1 in Escherichia coli and TEF1 in Saccharomyces cerevisiae as well as the inducible, oxygen-repressed promoter DAN1 in S. cerevisiae. Beyond generating promoter libraries, we discuss techniques to quantify the parameters of each new promoter. Promoter characteristics for each promoter in hand, the designer can then pick and choose the promoters needed for the specific genetic circuit described in silico. Second, Chemically Induced Chromosomal Evolution (CIChE) is presented as an alternative method to finely adjust RNA synthesis rates in E. coli by variation of gene cassette copy numbers in tandem gene arrays. Both techniques result in precisely defined RNA synthesis and should be of great utility in synthetic biology.

The σ subunit recognizes promoters

The enzymes that synthesize RNA on a DNA template are calledRNA polymerases. These enzymes do not initiate transcription randomly along the length of the chromosome. They start precisely where the gene starts. The transcriptional start sites are marked bypromoter sequences on the DNA, and the first task for the RNA polymerase is finding the promoter.

The RNA polymerase ofE. coli (Table 6.4) consists of a core enzyme with the subunit structure α2ββ′ω and a σ (sigma) subunit that is only loosely bound to the core enzyme.The σsubunit recognizes the promoter, and the core enzyme synthesizes RNA.

The promoters inE. coli have a length of about 60 base pairs, and they look quite different in different genes. Only two short segments, located about 10 base pairs and 35 base pairs upstream of the transcriptional start site, are similar in all promoters. Even these sequences are variable, although we can define aconsensus sequence of the most commonly encountered bases (Fig. 6.17).

This diversity is required because genes must be transcribed at different rates. Some are transcribed up to 10 times per minute, but others are transcribed only once every 10 to 20 minutes. The rate of transcriptional initiation depends on the base sequence of the promoter. In general,the more the promoter resembles the consensus sequence, the higher is the rate of transcription.

The RNA polymerase then separates the DNA double helix over a length of about 18 base pairs, starting at a conserved A-T–rich sequence about 10 base pairs upstream of the transcriptional start site. Strand separation is essential becausetranscription, like DNA replication, requires a single-stranded template.

The σ subunit separates from the core enzyme after the formation of the first 5 to 15 phosphodiester bonds. This marks the transition from the initiation phase to the elongation phase of transcription. The core enzyme now moves along the template strand of the gene while synthesizing the RNA transcript at a rate of about 50 nucleotides per second.

SUMMARY

RNA and protein synthesis have been studied in isolated surviving cod-islet tissue. Puromycin inhibited protein synthesis while having no affect upon the labelling of RNA. Actinomycin D stimulated both RNA and protein production. Glucose was found to have no affect upon either of these synthetic mechanisms.

Study of the incorporation of precursor into RNA over a time course of 120 min suggested that there are two phases of RNA synthesis.

Qualitative characterization of the labelled RNA demonstrated that it was of a molecular size similar to that of 3.5–4.0 S RNA. Recovered radioactivity from incubation of islet tissue with L-[Me-3H]-methionine was present in the low molecular weight RNA.

A comparison has been made of these piscine results with those from experiments conducted in a similar manner with mammalian islet tissue (rat).

From a consideration of these cod results, it is proposed that tRNA modulation may play a role in the regulation of the rate of proinsulin biosynthesis.

Viruses must first synthesize messenger RNA (mRNA)

Viruses contain either DNA or RNA, never both. The nucleic acids are present as single or double strands in a linear (DNA or RNA) or circular (DNA) form. The viral genome may be carried on a single molecule of nucleic acid or on several molecules. With these options, it is not surprising that the process of replication in the host cell is also diverse. In viruses containing DNA, mRNA can be formed using the host's own RNA polymerase to transcribe directly from the viral DNA. The RNA of viruses cannot be transcribed in this way, as host polymerases do not work from RNA. If transcription is necessary, the virus must provide its own polymerases. These may be carried in the nucleocapsid or may be synthesized after infection.

Making RNA

In bacteria, once the sigma subunit of RNA polymerase recognizes the −10 and −35 regions, the core enzyme forms a transcription bubble where the two DNA strands are separated from each other (Fig. 2.3). The strand used by RNA polymerase is called the template strand (aka noncoding or antisense) and is complementary to the resulting mRNA. The core enzyme adds RNA nucleotides in the 5′ to 3′ direction, based on the sequence of the template strand of DNA. The newly made RNA anneals to the template strand of the DNA via hydrogen bonds between base pairs. The opposite strand of DNA is called the coding strand (aka nontemplate or sense strand). Because this is complementary to the template strand, its sequence is identical to the RNA (except for the replacement of thymine with uracil in RNA).

RNA synthesis normally starts at a purine (normally an A) in the DNA that is flanked by two pyrimidines. The most typical start sequence is CAT, but sometimes the A is replaced with a G. The rate of elongation is about 40 nucleotides per second, which is much slower than replication (∼1000 bp/sec). RNA polymerase unwinds the DNA and creates positive supercoils as it travels down the DNA strand. Behind RNA polymerase, the DNA is partially unwound and has surplus negative supercoils. DNA gyrase and topoisomerase I either insert or remove negative supercoils, respectively, returning the DNA back to its normal level of supercoiling (see Chapter 4).

RNA polymerase makes a copy of the gene using the noncoding or template strand of DNA. RNA has uracils instead of thymines.

Transcription: Basic Mechanism

RNA synthesis requires base-pairing interactions between the single-stranded DNA template and ribonucleoside-5′-triphosphate (NTP) substrates. Thus, transcription involves unwinding of a small section (10–13 nucleotide residues) of the DNA double helix at which point one of the two DNA strands acts as a template for RNA synthesis. The unwound portion of DNA is called the transcription bubble; it contains a DNA–RNA hybrid of 8–9 base pairs. Each elementary step of transcription constitutes the incorporation of an NTP at the 3′ terminus of the growing RNA chain, so that the bubble propagates stepwise along the template accompanied by unwinding of the DNA duplex ahead, peeling of the transcript from the hybrid, and closing of the DNA duplex behind.

The nucleotide addition is accomplished through the formation of a phosphodiester bond and the release of pyrophosphate (PPi). It is a highly energetically favorable reaction (ΔG≅7kcal/mol), which is practically irreversible under physiological conditions but may be reversed in vitro by excess of PPi. As the result, a linear RNA polymer is built in the 5′ to 3′ direction. Selection of the substrates is achieved through hydrogen bonding between complementary bases, adenine to thymine (or uracil), and guanine to cytosine.

The enzyme that performs transcription of cellular DNA is called DNA-dependent RNA polymerases (RNAPs). The best characterized RNAP is that from the bacterium Escherichia coli. RNAP's catalytic core enzyme contains five subunits (α2, β, β′, and ω) and has an Mr of 390 kDa. The sixth interchangeable subunit, σ, is derived from a group of related polypeptides of different size. The σ-subunit is a specificity factor, which transiently associates with the core enzyme to form the holoenzyme that is capable of recognition of and specific binding to the promoter. The most common σ-factor in the bacterial cell is σ70. In contrast to DNA polymerase, the RNAP holoenzyme is able to initiate RNA synthesis starting from NTP and does not require a primer. Transcription is completely processive, i.e., RNAP is associated with the transcription bubble at all times in a ternary complex in which the nucleic acid scaffold slides through the protein as the bubble propagates. At the termination site, the ternary complex dissociates, the bubble collapses, and single-stranded RNA product is released.

Thus, synthesis of a particular transcript can be divided into three distinct steps: initiation, elongation, and termination, which jointly constitute the transcription cycle (Figure 1). Each step of the transcription cycle is subject to regulation either by factors external to the transcriptional apparatus or by signals encoded in the DNA sequence.

Effect of Cortisol on Polyribosomal RNA Synthesis

RNA synthesis was followed by the incorporation of 14C-uridine into the cultures during 5 h (Fig. 4). The fractionation procedure was identical to that described in the legend to Figure 3.

The absorbance profile of the polysomes from cortisol-treated tissue is identical to that of the control, however, the distribution of the labeled RNA accumulated during the 5 h in culture is different. Elevated radioactivity was found in the fractions from the cortisol-induced tissue which correspond to the region of increased translational activity seen in Figure 3. Analysis of the RNA extracted from these polysomes (dashed area in Fig. 4), by electrophoresis in polyacrylamide gels (Fig. 5a) shows that there is no detectable change in the synthesis of ribosomal RNA in response to Cortisol. The amount of label in the t-RNA molecules which are attached to the active polysomes, is very low, and there are no significant differences between the Cortisol induced and the control cultures. The difference in the level of labeled RNA in these polysomes, in response to Cortisol, may therefore be due to mRNA molecules that have accumulated in these fractions during the first hours of GS induction. The difference between induced and non-induced tissue in mRNA content of polysomes is shown in Figure 5b (Senitzki, 1972). The increase of this specific mRNA in response to Cortisol can also be detected in analyses of the entire cytoplasmic RNA, as shown in Figure 5 a. In addition, we find in the total cytoplasmic RNA of the Cortisol-induced retinas, an increase in some smaller informational molecules. Similar results were reported by Schwartz (1972).

Analysis of the total cytoplasmic RNA emphasizes again the similarity in the rate of r-RNA synthesis in the induced and non-induced neural retinas, as also in the rate of synthesis of t-RNA molecules.

Introduction and Background

RNA synthesis by DNA-dependent RNA polymerases (RNAPs) is processive, requiring a single enzyme molecule to transcribe the full length of a gene regardless of the length. The requirement for RNAP to remain resolutely associated with the DNA template through multiple kilobases necessitates an extremely stable transcription elongation complex that can transcribe through different sequences and protein-bound DNA templates. Despite this stability, cells must be able to halt RNA synthesis after transcription of a complete gene or operon, and stop any RNAP that has initiated transcription aberrantly. Failure to terminate transcription of an upstream gene could allow regulation-independent expression of downstream genes, and synthesis of untranslated or antisense transcripts with detrimental consequences; aberrant transcription is particularly problematic for the gene-dense chromosomes common to Bacteria and Archaea. Two general mechanisms have evolved to efficiently disrupt transcription elongation complexes that release the RNA transcript and recycle RNAP for further rounds of transcription.

Multi-subunit RNAPs from each domain share a near identical core structure that envelopes an 8- or 9-bp RNA:DNA hybrid within a tight-fitting pocket (Figure 1). High-resolution crystal structures and a wealth of biochemical data from many different RNAPs demonstrate that hydrogen bonding within the hybrid and contacts between the enclosed nucleic acids and RNAP provide stability to transcription elongation complexes. Despite similar transcription elongation complex architecture, RNAPs from different domains, and each of the eukaryotic RNAPs, respond to different termination signals and factors, suggesting that several mechanisms of transcription termination are possible, or that a diverse set of factors and sequences use a common mechanism to disrupt the complex. Conserved elongation factors (i.e., NusG and NusA) modify RNAP activities and add an additional level of regulation to the elongation–termination decision. The mechanistic details of transcript release are best understood in Bacteria, although some features are shared in each domain. This article focuses on transcription termination and its regulation in Bacteria, with relevant comments to bring attention to similarities and differences in Archaea and Eukarya.

2.1.5 Nucleotide pool and DNA damage response

RNA and DNA synthesis are increased in proliferating cells, especially in chronically proliferative cancer cells. As a result, maintenance of the nucleotide pool is critical for cancer cells to sustain their proliferative ability. It has been shown that cancer cells tend to use de novo nucleotide synthesis pathways, which require glycolysis metabolites, glutamine and aspartate. Deficiency in these metabolites causes increased radio-sensitivity (Villa et al., 2019). Importantly, ribose-5-phosphate from PPP provides the ribose backbone for DNA stability. For instance, deficiency of in PPP enzymes such as transketolase (TKT), transketolase-like 1 (TKTL1), and 6-phosphogluconate dehydrogenase (6PGDH) affects radio- and chemoresistance (Dong and Wang, 2017; Li et al., 2019; Liu et al., 2019). Additionally, N10-formyl-tetrahydrofolate (THF) from the folate cycle is necessary for purine ring synthesis, which is affected by the serine pathway (Villa et al., 2019). The upregulated glucose metabolism in cancer cells favors the production of above metabolites and supports the nucleotide pool (Hay, 2016). Taken together, these findings support the notion that nucleotide pool significantly affects the metabolic pathways involved in DNA damage responses.