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Monday, March 19, 2007

Gene and Transcription Orientation

The DNA double helix consists of two strands of DNA wound around each other to form the classic helical structure. One of the most important insights into solving the structure was when Watson and Crick realized that the two strands had to run in opposite direction. The ends of each strand are identified by the carbon atom on the deoxyribose sugar. One end is called the 5′ (five prime) end because the 5′ carbon atom is exposed. The other end is called the 3′ (three prime) end because the 3′ carbon atom is exposed.

RNA (and DNA) can only be synthesized from the 5′ to the 3′ direction. What this means is that at the beginning of the gene when the transcription bubble forms it's the template strand that's copied into RNA and the beginning of the template strand is the 3′ end. (It's the opposite orientation of the newly synthesized RNA.) [see Transcription]

The complementary strand of DNA is called the coding strand because it represents the sequence of the gene product. In other words, it's the same sequence as the RNA. By convention the orientation of the gene is determined by the coding strand and not the template strand. Thus, the beginning of a gene is called the 5′ end and the end of a gene is the 3′ end;.

The electron micrograph below shows E. coli ribosomal RNA genes being transcribed. The thin line (upper right) is the Double-stranded DNA strand. Transcription of the genes begins at the initiation site (lower left). This is the 5′ end of the genes.

RNA polymerase first bound to the initiation site and began transcribing in the 5′ to 3′ direction as shown. As the transcription complex moves along the gene the RNA product gets longer. In this case it is bound to protein so it looks compact. About halfway along the genes the RNA is processed by cutting and that's why it seems to get shorter near the middle of the gene.

The large ribosomal RNA is in the second half of the transcribed region. You can see that the RNA in the second half is larger than the small ribosomal RNA in the first half.

Note that there are many transcription complexes transcribing this region at the same time. In fact, they are about as closely packed as they can possibly be. These genes are being transcribed at the maximum possible rate. They have a very strong promoter.


Anonymous said...

Larry, I've always has a little trouble grasping Transcription. The one thing I never understand is what is a primer? Can you help?

Larry Moran said...

In the case of natural transcription it refers to the fact that you need to get the reaction started before entering the normal elongation mode.

During elongation the enzyme adds nucleotides on to a growing chain. This is a typical polymerization reaction but like all such reactons in biological systems you need to do something special to get the reaction started in the first place.

The starting bit of the polymer is called the "primer" because it primes the subsequent reactions. In the case of transcription it is RNA polymerase itself that makes the primer. (In other polymerization reactions it's often a seprate enzyme.)

The initiation complex makes a short primer of about 10 nucleotides while it is still bound to the promoter in the initiation stage. At that point it rearranges and shifts to the elongation stage. The reactions that make the primer are slower than the addition reactions and they require the initiation factors. We don't know exactly how the primer reaction works.

Anonymous said...

That's why I kept getting confused and annoying my lecturer. Sometimes it was referred to as the starting bit of the polymer and other times as the polymerase enzyme. Thank You!

Anonymous said...

hello Professor Larry, Can you tell me after cloning the cDNA in vector, what methods can we use to check that the gene is in correct orientation?


ajay said...

prof.Larry can each gene segment in a DNA can have different transcriptional orientation?

Anonymous said...

Hello prof. Is there any database from which we can retrieve different orientations ie convergent divergent and unidirectional gene pairs and its spacers?

Justin Hart said...

Thank you so much for these details! My question is this: at SNPedia and at Promethease they will take my raw data (from AncentryDNA) and reverse it based on orientation changes from new "build" (I think I have that right)... but what determines when they make that change? Is it the based on the comparison study using a different build? Then I upload it to impute.met they don't do the orientation changes and so I get different results.

Unknown said...

Justin - here at SNPedia, we have to take both the dbSNP and genome assembly builds into account, and then compare that to the build used by whichever company produced your raw data, in order to get the orientation correct. It's a lot of work.