Transcription and RNA Processing

Transfer of Genetic Information: The Central Dogma

DNA is found mostly in the nucleus. RNA is common in the cytoplasm. Watson and Crick proposed that RNA must copy the DNA message in the nucleus and carry it out to the cytoplasm, where proteins are synthesized. Crick predicted the existence of adaptor molecules that reads the genetic code and selects the appropriate amino acids to add to a growing polypeptide chain. The Central Dogma

The Central Dogma

The central dogma of biology is that information stored in DNA is transferred to RNA molecules during transcription and to proteins during translation.

The Central Dogma

Transcription involves the synthesis of an RNA transcript complementary to one strand of DNA of a gene. Translation is the conversion of information stored in the sequence of nucleotides in the RNA transcript into the sequence of amino acids in the polypeptide gene product, according to the specifications of the genetic code.

The Central Dogma

Transcription and Translation in Prokaryotes

The primary transcript is equivalent to the mRNA molecule. The mRNA codons on the mRNA are translated into an amino acid sequence by the ribosomes.

Transcription and Translation in Eukaryotes

The primary transcript (pre-mRNA) is a precursor to the mRNA. The pre-mRNA is modified at both ends, and introns are removed to produce the mRNA. After processing, the mRNA is exported to the cytoplasm for translation by ribosomes.

Types of RNA Molecules

Messenger RNAs (mRNAs)intermediates that carry genetic information from DNA to the ribosomes. Transfer RNAs (tRNAs)adaptors between amino acids and the codons in mRNA. Ribosomal RNAs (rRNAs)structural and catalytic components of ribosomes. Small nuclear RNAs (snRNAs)structural components of spliceosomes. Micro RNAs (miRNAs)short single-stranded RNAs that block expression of complementary mRNAs.

The Process of Gene Expression

Information stored in the nucleotide sequences of genes is translated into the amino acid sequences of proteins through unstable intermediaries called messenger RNAs.

General Features of RNA Synthesis

Similar to DNA Synthesis except
The precursors are ribonucleoside triphosphates. Only one strand of DNA is used as a template. RNA chains can be initiated de novo (no primer required).

The RNA molecule will be complementary to the DNA template (antisense) strand and identical to the DNA nontemplate (sense) strand (except that uridine replaces thymidine). RNA synthesis is catalyzed by RNA polymerases and proceeds in the 5 3 direction.

Template: This strand provides the pattern for transcription. (antisense) Nontemplate: This strand is the original message thats actually being transcribed (sense).

The Transcription Unit in Eukaryotes

The transcription unit is made up of promoter, gene, and terminator. The presence of TATA tells the transcription-starting enzyme that the gene to transcribe is about 30 to 50 base pairs away and also locates the template strand..

Transcription Promoter Sequences

RNA polymerase recognizes a specific base sequence in the DNA called a promoter and binds to it. Many eukaryotic promoters contain a TATA box (sequence TATAAA, often within 50 bases of the start site), where a TATA binding protein binds assisting in the formation of the RNA polymerase transcriptional complex.

Promoter: Technical Definition

A promoter is defined as the region upstream of a gene containing the binding site for RNA polymerase II that initiates transcription of the DNA. It contains a TATA box, a CAAT box or an AGGA box, and the CAP site.

Stages of Transcription

The early stages of transcription in prokaryotes, showing the components of the process.

Initiation of RNA Chains

1. Binding of RNA polymerase holoenzyme to a promoter region in DNA 2. Localized unwinding of the two strands of DNA by RNA polymerase to provide a single-stranded template 3. Formation of phosphodiester bonds between the first few ribonucleotides in the nascent RNA chain

The early stages of transcription in prokaryotes, showing template binding at the -10 site involving the subunit of RNA polymerase and subsequent initiation of RNA synthesis.

The early stages of transcription in prokaryotes, showing chain elongation, after the subunit has dissociated from the transcription complex and the enzyme moves along the DNA template.

Elongation

Termination Signals in E. coli

Rho-dependent terminatorsrequire a protein factor () Rho-independent terminatorsdo not require

Transcription Terminator Sequence (Prokaryotes)

1. Rho-independent transcription termination
involves sequences within the RNA (poly U residues) that signal the RNA polymerase to stop. The terminator sequences form a stem-loop hairpin structure that leads to the dissociation of the RNAP from the DNA template.

2. Rho-dependent termination - uses a termination factor called factor (rho factor) which is a protein to stop RNA synthesis at specific sites. Rho protein binds at a rho utilisation site on the RNA strand and runs along the mRNA towards the RNAP. A stem loop structure upstream of the terminator region pauses the RNAP, when -factor reaches the RNAP, it causes RNAP to dissociate from the DNA, terminating transcription.

Transcription Terminator Sequence (Eukaryotes)

Eukaryotic genes contain a poly-A signal located downstream of the last exon. This signal is used to add a series of adenylate residues during RNA processing. Transcription often terminates at 0.5 - 2 kb downstream of the poly-A signal, but the mechanism is unclear.

Transcription in Eukaryotes
Three different enzymes catalyze transcription in eukaryotes, and the resulting RNA transcripts undergo three important modifications, including the excision of noncoding sequences called introns. The nucleotide sequenced of some RNA transcripts are modified posttranscriptionally by RNA editing.

RNA Processing in Eukaryotes

Eukaryotic gene transcripts usually undergo three major modifications:
(1) the addition of 7-methyl guanosine caps to 5 termini, (2) The addition of poly(A) tails to 3 ends, and (3) Editing of the RNA sequence (4) The excision of noncoding intron sequences.

The 3 Poly(A) Tail

RNA Editing
Usually the genetic information is not altered in the mRNA intermediary. Sometimes RNA editing changes the information content of genes by
Changing the structures of individual bases Inserting or deleting uridine monophosphate residues.

Interrupted Genes in Eukaryotes: Exons and Introns

Most eukaryotic genes contain noncoding sequences called introns that interrupt the coding sequences, or exons. The introns are excised from the RNA transcripts prior to their transport to the cytoplasm.

Introns
Introns (or intervening sequences) are noncoding sequences located between coding sequences. Introns are removed from the pre-mRNA and are not present in the mRNA. Exons (both coding and noncoding sequences) are composed of the sequences that remain in the mature mRNA after splicing. Introns are variable in size and may be very large.

Types of Intron Excision

The introns of tRNA precursors are excised by precise endonucleolytic cleavage and ligation reactions catalyzed by special splicing endonuclease and ligase activities. The introns of some rRNA precursors are removed autocatalytically in a unique reaction mediated by the RNA molecule itself. The introns of nuclear pre-mRNA transcripts are spliced out in two-step reactions carried out by spliceosomes.

Excision of Intron Sequences

Splicing mechanism of pre-rRNA involving group I introns that are removed from the initial transcript. The process is one of self-excision involving two transesterification reactions.

Introns can be spliced out leaving all the exons in their original order, or introns and exons can be spliced out to create a new sequence of exons (also called alternative splicing). Alternative splicing results in the possibility for one gene to be expressed in different ways.