I. MICROBIAL GENETICS

E. POLYPEPTIDE AND PROTEIN SYNTHESIS

2. Transcription

Fundamental statements for this learning object:

1. During protein synthesis, the order of nucleotide bases along a gene gets transcribed into a complementary strand of mRNA which is then translated by tRNA into the correct order of amino acids for that polypeptide or protein.
2. The order of deoxyribonucleotide bases along the DNA determines the order of amino acids in the proteins, that is, its primary structure.
3. Because certain amino acids can interact with other amino acids, the order of amino acids for each protein determines its final three-dimensional shape, which in turn determines the function of that protein
.
4. Messenger RNA (mRNA) is synthesized by complementary base pairing of ribonucleotides with deoxyribonucleotides to match a portion of one strand of DNA called a gene.
5.Although genes are present on both strands of DNA, only one strand is transcribed for any given gene.
6.The enzyme RNA polymerase transcribes DNA.
7. To initiate transcription in bacteria, a variety of proteins called sigma factors bind to RNA polymerases. This complex can then bind to a specific DNA sequence called the promoter located along the DNA prior to the coding region of the gene. The promotor determines what region of the DNA and which strand of DNA will be transcribed into RNA.
8.Like DNA polymerase, RNA polymerase can only synthesize nucleic acid in a 5' to 3' direction while "reading" a DNA template in the 3' to 5' direction.
7. Once the RNA polymerase/sigma factor complex recognizes the correct promoter, the sigma factor dissociate from the RNA polymerase and the enzyme begins to unwind the helix of the DNA creating a region of nonpaired deoxyribonucleotides that serve as a template for RNA synthesis.
8. During transcription, ribonucleotides hydrogen bond through the process of complementary base pairing with the exposed deoxyribonucleotides on the unwound strand that is to be transcribed. The ribonucleotides are then covalently bonded together by phosphodiester bonds.
9.
This process continues until the RNA polymerase encounters a "stop" signal or transcription terminator at the end of the gene.
10. A single gene can be transcribed multiple times.
11. The mRNA molecule is divided up into codons. A codon is a series of three consecutive mRNA bases coding for one specific amino acid.
12.
Three codons, UAG, UAA, and UGA, function as stop or nonsense codons to terminate translation.
13. In bacteria, a mRNA can be monocistronic or polycistronic. A monocistronic mRNA is a transcript of a single gene; a polycistronic mRNA carries a transcript of multiple genes, often involved in a single biochemical pathway. Once transcribed, the mRNA can be translated into protein by tRNA on 70S ribosomes (composed of 50S and 30S subunits).
14. In bacteria, transcription and translation are coupled.
15. Transcription is more complex in eukaryotic cells than in those that are prokaryotic. Activator proteins bind to genes known as enhancers which help determine which genes are switched on and speed up transcription. Repressor proteins bind to genes called silencers which interfere with activator proteins and slow down transcription. Coactivators, adapter molecules which coordinate signals from activator and repressor proteins, relay this information to basal factors which then position RNA polymerase at the start of the coding region of the gene to begin transcription.
16. Most genes in higher eukaryotic cells contain regions called introns that are not part of the code for the final protein. These are interspersed among the coding regions or exons that actually code for the final protein.
17. After transcription of the precursor mRNA, non-protein coding regions (introns) are excised and coding regions (exons) are joined together by complexes of ribonucleoproteins called spliceosomes to produce what is termed mature mRNA.
18.
The mature mRNA then passes through the pores in the nuclear membrane to be translated into protein by tRNA on 80S ribosomes (composed of 60S and 40S subunits) in a manner similar to prokaryotes.

 

LEARNING OBJECTIVES FOR THIS SECTION


DNA is divided into functional units called genes. A gene (def) is a segment of DNA that codes for a functional product (mRNA, tRNA, or rRNA). Since the vast majority of genes are transcribed into mRNA and mRNA is subsequently translated into polypeptides or proteins, most genes code for protein synthesis. The term polypeptide (def) refers to many amino acids (def) connected by peptide bonds (def). While all proteins are polypeptides, not all polypeptides are proteins. In some cases, smaller polypeptides coded for by two or more genes must be joined together to produce a functional protein. In other cases, as will be mentioned below, mRNA carries a transcript of several genes resulting in the synthesis of a large polypeptide that must subsequently be cleaved by enzymes called proteases into two or more smaller functional proteins. For simplicity, we will use the term protein when referring to the end product of transcription and translation. In this section we will see how the sequence of deoxyribonucleotide bases along one strand of DNA ultimately codes for the amino acid sequence of a particular polypeptide or protein.

During protein synthesis, the order of nucleotide bases along a gene gets transcribed into a complementary strand of mRNA which is then translated by tRNA into the correct order of amino acids for that polypeptide or protein. Therefore, the order of deoxyribonucleotide bases (def) along the DNA determines the order of amino acids in the proteins, that is, its primary structure (def). Because certain amino acids can interact with other amino acids, the order of amino acids for each protein determines its final three-dimensional shape, which in turn determines the function of that protein.

Protein synthesis can be divided into two stages: transcription and translation. In this section we will look at transcription.


Transcription (def)

1. Transcription in Prokaryotic Cells

Description: Messenger RNA (mRNA) (def) is synthesized by complementary base pairing (def) of ribonucleotides (def) with deoxyribonucleotides (def) to match a portion of one strand of DNA called a gene (def). Although genes are present on both strands of DNA, only one strand is transcribed for any given gene. Following transcription, 30S and 50S ribosomal subunits attach to the mRNA and tRNA inserts the correct amino acids which are subsequently joined to form a polypeptide or a protein through a process called translation.

The enzyme RNA polymerase (def) transcribes DNA. This enzyme initiates transcription, joins the RNA nucleotides together, and terminates transcription. To initiate transcription in bacteria, a variety of proteins called sigma factors (def) bind to RNA polymerases. This complex can then bind to a specific sequence of usually about 40 deoxyribonucleotide bases called the promoter (def) located along the DNA prior to the coding region of the gene. The promotor determines what region of the DNA and which strand of DNA will be transcribed into RNA.

Like DNA polymerase, RNA polymerase can only synthesize nucleic acid in a 5' to 3' direction while "reading" a DNA template in the 3' to 5' direction. As mentioned earlier in this unit, the 3' end (def) of a strand of nucleic acid has a hydroxyl (OH) group on the 3' carbon of the deoxyribose or ribose and is not linked to another nucleotide. The 5' end (def) of that strand has a phosphate group attached to the 5' carbon of the sugar and is not linked to another nucleotide (see Fig. 1).

Once the RNA polymerase/sigma factor complex recognizes the correct promoter, the sigma factor dissociate from the RNA polymerase and the enzyme begins to unwind the helix of the DNA creating a region of nonpaired deoxyribonucleotides that serve as a template for RNA synthesis (see Figs. 2 and 3).

GIF animation illustrating RNA polymerase unwinding the DNA helix during transcription

While the RNA polymerase does not transcribe the promoter itself, it does transcribe a short noncoding leader sequence (def) just prior to the coding sequence (def) of the gene. The leader sequence is the portion of DNA that is transcribed into the ribosome-binding site of the mRNA (see below under translation.) The coding sequence contains the actual message for protein synthesis.

Once the actual transcription begins, ribonucleotides containing 3 phosphate groups hydrogen bond through the process of complementary base (def) pairing with the exposed deoxyribonucleotides on the unwound strand that is to be transcribed (see Fig. 4). The ribonucleotides are then covalently bonded together by phosphodiester bonds (def), the energy being supplied by the cleavage of two phosphate groups from the ribonucleotide triphosphate (see Fig. 5). (The phosphodiester bond refers to the phosphate on the 5'C of the newly inserted nucleotide covalently bonding to the 3'C of the last ribonucleotide in the mRNA chain.) The mRNA polymerizes at a rate of about 30 nucleotides per second.

GIF animation illustrating the synthesis of mRNA complementary to DNA during transcription

As the RNA polymerase moves down the DNA, the previous stretch of DNA again pairs with its complementary strand. This process continues until the RNA polymerase encounters a "stop" signal or transcription terminator (def) at the end of the gene. This causes the completed mRNA to drop off the gene.

Once the RNA polymerase moves beyond the promotor region, a new molecule of RNA polymerase can bind to the promotor and start a new round of transcription. In this way, a single gene can be transcribed multiple times.

 

 

Transcription is summarized in Figs. 6 and 7.

There are 22 amino acids that can be encoded by the genetic information carried on mRNA. The mRNA molecule is divided up into codons. A codon (def) is a series of three consecutive mRNA bases coding for one specific amino acid. The various codons and the amino acids for which they code are shown in Fig. 8. There are 64 codons. One codon, AUG, also serves as a start codon (def) to initiate translation, and three codons, UAG, UAA, and UGA, function as stop or nonsense codons (def) to terminate translation. (Alternative start codons are different from the standard AUG codon and are found occasionally in both prokaryotes and eukaryotes.)

In bacteria, a mRNA can be monocistronic (def) or polycistronic (def). A monocistronic mRNA is a transcript of a single gene. A polycistronic mRNA carries a transcript of multiple genes, often involved in a single biochemical pathway. Groups of related genes that are transcribed together to form a polycistronic mRNA are known as operons. There are also specific genes along the DNA from which each of the different transfer RNAs (tRNAs) (def) and the ribosomal RNAs (rRNAs) (def) are transcribed.

Most mRNAs in prokaryotes have a half-life on the order of a few minutes. Molecules of rRNA and tRNA, on the other hand, are much more stabile. Because rRNA and tRNA are highly folded molecules, unlike mRNA, they are much more resistant to degradation by ribonucleases.

In bacteria, transcription and translation (def) are coupled. RNA polymerase (def) binds to the 30S ribosomal subunit of prokaryotic ribosomes to form a transcription and translation machine called an expressome. As the DNA is being unwound and transcribed into complementary mRNA by RNA polymerase, the mRNA is being fed into the translational center of the ribosome where it is being translated into a polypeptide. Translation is described in the next lesson.   

Coupled transcription and translation in bacteria via the bacterial expressome.
Science News.

 

 

2. Transcription in Eukaryotic Cells

Transcription (def) is more complex in eukaryotic cells than in those that are prokaryotic. Activator proteins (def) bind to genes known as enhancers (def) which help determine which genes are switched on and speed up transcription. Repressor proteins (def) bind to genes called silencers (def) which interfere with activator proteins and slow down transcription. Coactivators (def), adapter molecules which coordinate signals from activator and repressor proteins, relay this information to basal factors (def) which then position RNA polymerase (def) at the start of the coding region of the gene to begin transcription.

Once the actual transcription begins, ribonucleotides containing 3 phosphate groups form hydrogen bonds through the process of complementary base (def) pairing with the exposed deoxyribonucleotides on the unwound strand that is to be transcribed. The ribonucleotides are then covalently bonded together by phosphodiester bonds (def), the energy being supplied by the cleavage of two phosphate groups from the ribonucleotide triphosphate (see Fig. 5). (The phosphodiester bond refers to the phosphate on the 5'C of the newly inserted nucleotide covalently bonding to the 3'C of the last ribonucleotide in the mRNA chain.)

Unlike prokaryotes, most genes in higher eukaryotic cells contain large amounts - as much as 98% in the human genome - of regions called introns (def) that are not part of the code for the final protein. These are interspersed among the coding regions or exons (def) that actually code for the final protein.

RNA polymerase copies both the exons and the introns to form what is called precursor mRNA or pre-mRNA (def). Early in transcription, a cap in the form of an unusual nucleotide, 7-methylguanylate, is added to the 5' end of the pre-mRNA. This cap helps ribosomes attach for translation (def). As transcription is nearly completed, a series of 100-250 adenine ribonucleotides called a poly-A tail is added to the 3' end of the pre-mRNA. This poly-A tail is thought to help transport the mRNA out of the nucleus and may stabilize the mRNA against degradation in the cytoplasm. After transcription of the precursor mRNA, non-protein coding regions (introns) are excised and coding regions (exons) are joined together by complexes of ribonucleoproteins called spliceosomes to produce what is termed mature mRNA (def) as shown in Fig. 9. This process is called RNA processing (def).

 

 

The mature mRNA then passes through the pores in the nuclear membrane to be translated (def) into protein by tRNA (def) on eukaryotic 80S (def) ribosomes (def) (composed of 60S and 40S subunits) in a manner similar to prokaryotes.

The mRNA molecule is divided up into codons. A codon (def) is a series of three consecutive mRNA bases coding for one specific amino acid. The various codons and the amino acids for which they code are shown in Fig. 8. There are 64 codons. One codon, AUG, also serves as a start codon (def) to initiate translation, and three codons, UAG, UAA, and UGA, function as stop or nonsense codons (def) to terminate translation. (Alternative start codons are different from the standard AUG codon and are found occasionally in both prokaryotes and eukaryotes.)

In addition to the genes that are transcribed into mRNA to be translated into polypeptides and proteins, there are also specific genes in the DNA from which each of the different transfer RNAs (tRNAs) (def) and the ribosomal RNAs (rRNAs) (def) are transcribed.

Once transcribed, the mRNA can be translated into protein.

As mentioned above, introns make up the majority of DNA in higher eukaryotic cells and for decades was considered to be "junk DNA" accumulated over millions of years of evolution. Over recent years however, it has been discovered that much of this intergenic DNA, although it does not code for protein synthesis, is transcribed into functional molecules of RNA with names such as antisense RNA microRNA, and riboswitch RNA that play important roles in whether or not a protein is actually made.

Antisense RNA is RNA transcribed off of the strand of DNA complementary to the one being transcribed into mRNA. In other words, it is an RNA molecule complementary to a mRNA and as such may complementary base pair with the mRNA and prevents it from being translated into protein.

MicroRNA, often transcribed from intron DNA, folds over upon itself to resemble double-stranded RNA, a form of RNA produced by many viruses during their life cycle. Viral double-stranded RNA activates a host defense mechanism that degrades that viral RNA. The MicroRNA frequently binds to mRNA and tricks this defense mechanism into degrading that mRNA so it can not be translated into protein.

Riboswitch RNA, often transcribed from introns, exists in an inactive form until a specific target chemical binds. The binding of the target chemical turns the riboswitch RNA to an active form that can be translated into a specific protein.


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