The genetic code is often called the “master plan” because it contains the instructions a cell needs to maintain itself.
We now know there is more to these instructions than simply the sequence of letters in the nucleotide code.
For example, large amounts of evidence show that this code is the basis for producing several molecules, including RNA and protein. The research has also shown that the instructions stored in the DNA are “read” in two steps: transcription and translation.
A portion of the double-stranded DNA template results in a single-stranded RNA molecule in transcription. In some cases, the RNA molecule is a “finished product” that plays a vital role within the cell.
Often, however, the transcription of an RNA molecule is followed by a translation step, which ultimately results in the production of a protein molecule.
An electron micrograph shows black threads of chromatin on a gray background. Chromatin threads are seen as thin vertical lines.
The horizontal lines branch off from the vertical lines to the left and the right; the horizontal lines look like the branches of a pine tree.
The dark black circular structures at the end of each branch are terminal knobs and contain RNA processing machinery.
The transcription process can be visualized by electron microscopy; this method was observed for the first time in 1970. In these early electron micrographs, the DNA molecules appear as “trunks,” with many “branches” of RNA that extend from them.
When DNAse and RNA (enzymes that degrade DNA and RNA, respectively) were added to the molecules, the application of DNAse eliminated the structures of the trunk. In contrast, the use of RNAse eliminated the branches.
DNA is double-stranded, but only one strand serves as a template for transcription at any given time. This template string is called the non-coding string. The non-stamping chain is called the coding chain because its sequence will be the same as that of the new RNA molecule.
In most organisms, the DNA strand that serves as a template for a gene may be the non-variable strand for other genes within the same chromosome.
The transcription process
The transcription process begins when an enzyme called RNA polymerase (RNA pol) binds to the DNA template strand and begins to catalyze the production of complementary RNA.
Polymerases are large enzymes composed of about a dozen subunits, and when they are active in DNA, they are also often complex with other factors. In many cases, these factors indicate which gene is transcribed.
Stages of transcription:
The first step in transcription is initiation when RNA pol binds to the current DNA of the gene in a specialized sequence called a promoter. In bacteria, promoters are generally composed of three sequence elements, while in eukaryotes, there are up to seven pieces.
In prokaryotes, most genes have a sequence called the Pribnow box. The TATA consensus sequence is about ten base pairs from the site that serves as the transcription initiation location.
Not all Pribnow boxes have this exact nucleotide sequence; these nucleotides are simply the most commonly found in each site. Although substitutions occur, each box is quite similar to this consensus.
Many genes also have the TTG CCA consensus sequence at 35 bases upstream from the start site. Some have an upstream element, an AT-rich region of 40 to 60 nucleotides upstream that increases the rate of transcription.
In any case, by joining, the “central enzyme” of the RNA pol binds to another subunit called the sigma subunit to form a holoenzyme capable of unwinding the double helix of the DNA to facilitate access to the gene.
The sigma subunit transmits the specificity of the promoter to the RNA polymerase; that is, it is responsible for telling the RNA polymerase where to join. Several different sigma subunits bind to other supporters and help activate and deactivate genes as conditions change.
Eukaryotic promoters are more complex than their prokaryotic counterparts, partly because eukaryotes have the three RNA above polymerase classes that transcribe different sets of genes.
Many eukaryotic genes also possess enhancer sequences, which can be found at considerable distances from their affected genes.
The enhancer sequences control the activation of the gene by binding to the activating proteins and altering the 3-D structure of the DNA to help “attract” the pol II RNA, thus regulating the transcription.
Because eukaryotic DNA is packaged tightly like chromatin, transcription also requires a series of specialized proteins that help make the template chain accessible.
The Rho-independent termination sequences stop transcription. The independent terminators of Rho contain inverted repeats followed by an adenine tail.
When the inverted repeats are transcribed at the end of an mRNA sequence, the inverted repeats can form a hairpin loop, which causes the RNA polymerase to stop transcription.
When the bonds break between the adenine-uracil base pairs in the adenine tail, the mRNA is released, and transcription is interrupted.
Repeated inverted sequences at the end of a gene allow folding of the newly transcribed RNA sequence into a hairpin loop. This completes the transcription and stimulates the release of the mRNA chain from the transcription machinery.
The terminator sequences are located near the ends of the non-coding lines. Bacteria possess two types of these sequences.
In the rho-independent terminators, the inverted repeat sequences are transcribed; then, they can bend over themselves in hairpin loops, causing RNA pol to stop and the transcript to be released.
On the other hand, the rho-dependent terminators use a factor called Rho, which actively unrolls the DNA-RNA hybrid formed during transcription, thus releasing the newly synthesized RNA.
In eukaryotes, transcription termination occurs by different processes, depending on the same polymerase used. For pol I genes, transcription is stopped using a termination factor through a mechanism similar to the rho-dependent termination in bacteria.
Transcription of pol III genes terminates after transcription of a termination sequence that includes a stretch of polyuracil by a mechanism that resembles the prokaryotic termination independent of Rho. However, the ending of pol II transcripts is more complex.
Transcription of pol II genes can continue for hundreds or even thousands of nucleotides beyond the end of a non-coding sequence. The RNA chain is then divided by a complex that seems to associate with the polymerase.
The excision seems to be coupled with transcription termination and occurs in a consensus sequence. Mature pol II mRNAs are polyadenylated at the 3 ‘end, resulting in a poly (A) tail; this process follows the division and is coordinated with the termination.
Both polyadenylation and termination use the same consensus sequence, and the interdependence of the processes was demonstrated in the late 1980s by the work of several groups.
A group of scientists working with mouse globin genes showed that the introduction of mutations in the AAT AAA consensus sequence, which is known to be necessary for adding poly (A), inhibited both polyadenylation and termination of transcription.
They measured the extension of the termination by hybridizing transcripts with the different poly (A) consensus sequence mutants with wild-type transcripts and could see a decrease in the hybridization signal, suggesting that appropriate termination was inhibited.
Therefore, they concluded that polyadenylation was necessary for the termination (Logan et al., 1987).
Another group obtained similar results using a monkey viral system, SV40 (simian virus 40). They introduced mutations in a poly (A) site, which caused the mRNAs to accumulate well above the wild type (Connelly & Manley, 1988).
Excision ratio and termination
The exact relationship between excision and termination has not yet been determined.
A model assumes that the split triggers the termination; another proposes that the polymerase activity is affected when it passes through the consensus sequence at the cut-off site, perhaps through changes in the associated transcriptional activation factors.