Principles of Cell Biology (BIOL2060)

Department of Biology
Memorial University of Newfoundland

Transcription

The Central Dogma of Molecular Biology is really an informational flow chart.

DNA synthesis       ->     RNA synthesis      ->     protein synthesis
(DNA replication)            (transcription)                  (translation)

DNA synthesis maintains the genetic information and passes this to the next generation.
RNA synthesis (transcription) is a transfer of the information from the DNA where it is stored into RNA which can be transported and interpreted.
The language (of nucleic acids) is essentially the same.
Messenger RNA (mRNA) moves the information from the DNA to the ribosomes to direct the production of protein.
Translation represents a change in the language from the nucleotide letters in RNA to the amino acid letters in protein.

All three major classes of RNA (mRNA, tRNA, &  rRNA) are synthesized by transcription of the appropriate genes and are involved in protein synthesis.
All three major types of RNA are involved in directing the formation of protein.
    1) mRNA carries the message from the DNA to the ribosome.
    2) rRNA’s are major structural components of the protein-synthesizing ribosome.
    3) tRNA’s act as adaptor molecules in aligning the amino acids according to the sequence present in the mRNA.

The genetic code is a system of purines and pyrimidines used to send messages from the genome to the ribosomes to direct protein synthesis.
A message written as a sequence of nucleotides in an mRNA molecule has no obvious meaning, until a set of equivalency rules for the genetic code is used to convert the sequence into the amino acid sequence of a recognizable polypeptide.

One strand of the DNA duplex (the template strand) is transcribed into a segment of mRNA shown, according to the same base-pairing rules used in DNA replication, except the base U is used in RNA in place of T.
The complementary DNA strand, with a sequence essentially identical to that of the mRNA, is called the coding strand.
With a nonoverlapping code, the reading frame advances three nucleotides at a time, and a mRNA segment is therefore read as three successive triplets, coding for amino acids.

The genetic code "words" are three-letter codons present in the nucleotide sequence of mRNA, as read in the 5prime to 3prime direction.
Each codon specifies either an amino acid or a stop signal.
To decode a codon, read down the left edge for the first letter, then across the grid for the second letter, and then down the right edge for the third letter.
For example, the codon AUG represents methionine. AUG is also the translational start signal.
There are 64 possible codons in mRNA, 61 code for amino acids.
TAA, TAG and TGA are the stop codons which do not have a corresponding tRNA.
The genetic code is universal (for the most part).

Transcription in Prokaryotic Cells

Transcription is catalyzed by RNA polymerase which makes RNA using DNA as a template.
Transcription of DNA occurs in four main stages:
1) binding of RNA polymerase to DNA at a promoter,
2) initiation of transcription on the template DNA strand,
3) subsequent elongation of the RNA chain, and
4) eventual termination of transcription, accompanied by the release of RNA polymerase and the completed RNA product from the DNA template.

RNA polymerase moves along the template strand of the DNA in the 3-prime to 5-prime direction, and the RNA molecule grows in the 5-prime to 3-prime direction.

The DNA promoter region in prokaryotes is a stretch of about 40 base pairs adjacent to and including the transcription startpoint.
By convention, the critical DNA sequences are given as they appear on the coding strand (the nontemplate strand, which corresponds in sequence to the RNA transcript).
The essential features of the promoter are the startpoint (designated +1 and usually an A), the six-nucleotide -10 sequence, and the six-nucleotide -35 sequence.
The two key sequences are located approximately 10 nucleotides and 35 nucleotides upstream from the startpoint.
The numbers of nucleotides separating the consensus sequences from each other and from the startpoint are important for promoter function, but the identity of these nucleotides is not.

During elongation, RNA polymerase binds to about 30 base pairs of DNA (each complete turn of the DNA double helix is about 10 base pairs).
At any given time, about 18 base pairs of DNA are unwound, and the most recently synthesized RNA is still hydrogen-bonded to the DNA, forming a short RNA-DNA hybrid.
This hybrid is probably about 12 base pairs long, but it may be shorter.
The total length of growing RNA bound to the enzyme and/or DNA is about 25 nucleotides.

Termination of transcription requires a termination sequence that triggers the end of transcription.
Two classes exist, rho dependent and rho independent.
In rho independent termination, a short complementary GC-rich sequence (followed by several U residues) will form a "brake" that will help release the RNA polymerase from the template (at the weakly poly-U stretch).
In rho dependent termination, binding of rho to the mRNA releases it from the template.

Transcription in Eukaryotic Cells

Although transcription in eukaryotes is similar to that in prockaryotes, the process appears to be complex.
Instead of one RNA polymerase, there are three (RNA Polymerases I, II, and III) involved in eukaryotic transcription.
RNA polymerase I (localized to the nucleolus) transcribes the rRNA precursor molecules.
RNA polymerase II produces most mRNAs and snRNAs.
RNA polymerase III is responbsible for the production of pre-tRNAs, 5SrRNA and other small RNAs.
The mitochondia and chloroplasts have their own RNA polymerases.

Eukaryotic nuclear genes have three classes of promoters which are individual for the three types of RNA polymerases
RNA polymerase I:  The promoter for RNA polymerase I has two components: 1) a core promoter (surrounding the startpoint) and 2) an upstream control element.
After the binding of appropriate transcription factors to both sites, RNA polymerase I binds to the core promoter.
RNA polymerase II:  The typical promoter for RNA polymerase II has a short initiator sequence, consisting mostly of pyrimidines and usually a TATA box about 25 bases upstream from the startpoint.
This type of promoter (with or without the TATA box) is often called a polymerase II core promoter, because for most genes a variety of upstream control elements also play important roles in the initiation of transcription.
RNA polymerase III:  The promoters for RNA polymerase III vary in structure but the ones for tRNA genes and 5S rRNA genes are located entirely downstream of the startpoint, within the transcribed sequence.
In tRNA genes, about 30-60 base-pairs of DNA separate promoter elements; in 5S rRNA genes, about 10-30 base-pairs promoter elements.

General transcription factors and the polymerase undergo a pattern of  sequential binding to initate transcription of nuclear genes.
1) TFIID binds to the TATA box followed by
2) the binding of TFIIA and TFIIB.
3) The resulting complex is now bound by the polymerase, to which TFIIF has already attached.
4) The initiation complex is completed by the addition of TFIIE, TFIIJ, and TFIIH.
5) After an activation step requiring ATP-dependent phosphorylation of the RNA polymerase molecule, the polymerase can initiate transcription at the startpoint.

The TATA-binding protein (TBP) is a subunit of the TFIID and plays a role in the activity of both RNA polymerase I and RNA polymerase III transcription.
TBP is also essential for transcription of TATA-less genes.
TBP differs from most DNA-binding proteins in that it interacts with the minor groove of DNA, rather than the major groove and imparts a sharp bend to the DNA.
The TBP has been highly conserved during evolution.
When TBP is bound to DNA, other transcription-factor proteins can interact with the convex surface of the TBP saddle.
TBP is required for transcription initiation on all types of eukaryotic promoters.

Termination signals end the transcription of RNA by RNA polymerase I and RNA polymerase III without the activity of hairpin structures as seen in prokaryotes.
mRNA is cleaved 10 to 35 base-pairs downstream of a AAUAAA sequence (which acts as a poly-A tail addition signal).

Ribosomal RNA processing involves cleavage of multiple rRNAs from a common precursor.
The eukaryotic transcription unit that includes the genes for the three largest rRNAs occurs in multiple copies and arranged in tandem arrays with nontranscribed spacers separate the units.
Each transcription unit includes the genes for the three rRNAs and transcribed spacer regions.
The transcription unit is transcribed by RNA polymerase I into a single long transcript (pre-rRNA) with a sedimentation coefficient of about 45S.
RNA processing yields mature rRNA molecules.
RNA cleavage actually occurs in a series of steps which varies in order with the species and cell type but the final products are always the same three types of rRNA molecules.

Every tRNA gene is transcribed as a precursor that must be processed into a mature tRNA molecule by the removal, addition and chemical modification of nucleotides.
Processing for some tRNA involves
    1) removal of the leader sequence at the 5 prime end
    2) replacement of two nucleotides at the 3 prime end by the sequence CCA (with which all mature tRNA molecules terminate)
    3) chemical modification of certain bases and
    4) excision of an intron.
The mature tRNA is often diagrammed as a flattened cloverleaf which clearly shows the base pairing between self-complementary stretches in the molecule.

Messenger RNA in eukaryotes is first made as heterogeneous nuclear mRNA (or pre-mRNA) then processed into mature mRNA through the addition of a 5 prime cap structure, addition of poly-A tails and the splicing out of introns.
To give the mRNA stability, a  5 prime "cap" (a guanosine nucleotide methylated at the 7th position) is joined to the 1st nucleotide in an unusual  "5 prime to 5 prime" linkage (sort of "backwards").
During the capping process, the first two nucleotides of the message may also become methylated

Transcription of eukaryotic pre-mRNAs often proceeds beyond the 3prime end of the mature mRNA.
An AAUAAA sequence located slightly upstream from the proper 3prime end then signals that the RNA chain should be cleaved about 10-35 nucleotides downstream from the signal site, followed by addition of a poly-A tail catalyzed by poly(A) polymerase.

Spliceosomes remove introns from pre-mRNA.
Introns were descovered to exist in eucaryotic mRNA by
    1) mixing mature mRNA molecules with the genes (DNA) from which they had been transcribed and
    2) examining the hydrogen bonded hybrids under an electron microscope.
Hybridization of a eukaryotic mRNA molecule with a gene which has one intron will produce two single-stranded DNA loops where the mRNA has hybridized to the DNA template strand plus an obvious double-stranded DNA loop.
The double-stranded DNA loop represents the intron, which contains sequences that do not appear in the final mRNA.
Restriction enzyme analysis has revealed the presence of introns.

The spliceosome is an RNA-protein complex that splices intron-containing pre-mRNA in the eukaryotic nucleus.
The substrate here is a molecule of pre-mRNA with two exons and one intron.
In a stepwise fashion, the pre-mRNA assembles with the U1 snRNP, U2 snRNP, and U4/U6 and U5 snRNPs (along with some non-snRNP splicing factors), forming a mature spliceosome.
The pre-mRNA is then cleaved at the 5prime splice site and the newly released 5prime end is linked to an adenine (A) nucleotide located at the branch-point sequence, creating a looped lariat structure.
Next the 3prime splice site is cleaved and the two ends of the exon are joined together, releasing the intron for subsequent degradation.
Alternative splicing results in alternate forms of mRNA and, often, proteins.

Most mRNA molecules have a high turn over rate as the molecules are rapidly degraded and replaced.
tRNA and rRNAs are relatively stable.
Bacterial mRNAís have half-lives of a few minutes and eukaryotic mRNA range from hours to days.
Transcription allows amplification of the genetic information because many copies of the mRNA can be produced to direct a great deal of protein synthesis.

Notes prepared from Becker's World of the Cell, 9th edition
Hardin & Bertoni, 2015
Figures copyright of Pearson Education Inc.
email me at bestave@mun.ca