Protein Synthesis Step 1- Transcription Overview

Every living cell contains instructions on how to synthesize all of the proteins needed to support life. These instructions are carried along strands of DNA^*. The process of synthesizing proteins from these instructions occurs over several steps. The first step is transcription.

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Video Overview

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During transcription, the enzyme RNA polymerase targets a short stretch of DNA containing just the information needed to synthesize one or more proteins, protein subunits, or other functional RNAs. The result of transcription is a single-stranded RNA molecule. Cells use transcription to synthesize all RNA, including transfer RNA (tRNA), ribosomal RNA (rRNA), and messenger RNA (mRNA).

[image transcriptionMakesRNA.png] All cellular RNA is synthesized by transcription.

For transcription to occur, a length of the double-stranded DNA needs to unwind to provide access to the bases to be transcribed.

Only one strand of the double strand of DNA is used to synthesize RNA. This strand is called the template strand. It is also called the non-coding strand. The strand that is not used is called the coding strand. The non-template strand is called the coding strand because the order of bases in the transcribed RNA match the order in the coding strand except that the RNA is made of RNA nucleotides^*, not DNA nucleotides.

The lengths of DNA used to synthesize individual proteins are relatively short compared to the overall length of an individual chromosome^*, but they are typically thousands of bases long.

• The stretch of DNA transcribed contains three regions.
• The upstream region holds the sequences of bases targeted by the cellular machinery to initiate transcription.
• The coding region has the information needed to synthesize the protein.
• The downstream region is where RNA synthesis stops.

Transcription is divided broadly into three phases.

The process starts with initiation - when the enzyme complex required for transcription binds to a promoter sequence upstream of the coding region.

Once transcription is initiated, it proceeds through Elongation. During this time, the bulk of the RNA is synthesized.

The process ends with termination in the downstream region due to the presence of DNA sequences that help to destabilize the enzyme complex’s attachment to the template strand, terminating transcription.

In prokaryotes, the enzyme responsible for RNA synthesis is RNA polymerase. This is referred to as the core enzyme. The core enzyme is fully capable of synthesizing RNA from a DNA template once it is bound to a DNA strand, but it does not readily bind to DNA without the help of other factors.

For initiation to occur, a protein called a sigma factor must first bind to the core enzyme. The complex formed when a sigma-factor binds to an RNA polymerase is called the holoenzyme. This is the biochemically active form of the polymerase capable of attaching to the promoter region and initiating transcription.

During initiation, the holoenzyme binds to a promoter through interactions between the sigma factor and specific sequences in the promoter. The transcriptional start site is the first base transcribed. It is given the label plus 1. The base just upstream of the start site is counted as negative 1, and numbers increase in the negative direction upstream.

Early studies of promoters found that the bases around -10 and -35 are conserved in many promoters. More recent work has identified additional conserved bases on either side of the original-10 region and further upstream between -40 and -60. Each of these regions is understood to play a role in how the holoenzyme engages with the promoter during initiation. The regions are called the -10 element, the -35 element, the extended -10 region, and the UP elements.

During initiation, the holoenzyme must bind to a promoter in the proper orientation and at the precise place required for the enzyme to begin RNA synthesis at the plus one base. This means the position and distance between the conserved regions are important.

Sigma factors control this positioning by binding to the template strand at the -10 and -35 elements and the extended -10 region, while the core RNA polymerase interacts with the UP elements.

The binding of the sigma factor helps separate the double-stranded DNA and orient the template strand so the core enzyme has access to the bases it uses as a template for RNA synthesis.

The fact that the core enzyme cannot initiate transcription without the aid of a sigma factor gives prokaryotes the ability to use sigma factors to regulate gene expression by controlling the types of sigma factors present in the cell and their relative abundance over time.

Additional levels of gene regulation are achieved through variations in the bases in the conserved regions. Some promoter regions have many bases in common with the consensus sequence, while others are not as well conserved. Each variation within the -10, -35, extend -10, and UP elements provide prokaryotes with additional opportunities to regulate gene expression.

RNA synthesis begins once the holoenzyme is bound to the DNA in the proper orientation and the double-strand has been separated around the transcriptional start site.

After the sigma factor has completed its role in initiation, it separates from the holoenzyme, and the core enzyme continues the elongation of the RNA strand on its own.

Elongation proceeds until the polymerase encounters a stop signal or terminator sequence.

Termination occurs in two ways - Rho-dependent termination and Rho-independent or intrinsic termination.

Rho-dependent termination depends on the involvement of an additional protein called a rho-protein, while intrinsic termination results from interactions between the RNA polymerase and the RNA strand being synthesized without the assistance of other factors. Both types of termination involve the formation of a hairpin loop in the RNA strand.

Loop formation results from lengths of complementary bases in the RNA strand that bind to each other, forming a loop. The loop is believed to interfere with the enzyme causing the rate of RNA synthesis to slow down and pause.

During Rho-dependent termination, the pause in transcription is thought to give the rho-protein time to bind to that region and destabilize the connection between the core enzyme and the template strand.

Termination sequences that depend on intrinsic termination contain a string of Uracil bases just downstream of the hairpin loop. This results in a stretch of relatively weak Adenine-uracil base pairings that appear to destabilize the enzyme complex without the involvement of a rho-protein.

In both cases, this destabilization causes the enzyme to fall off the DNA template, terminating transcription and releasing the completed single-stranded RNA. If the RNA is messenger RNA, it is available to act as a template for protein synthesis. If it is any other type of RNA, it is ready to be processed for its functional role in the cell.