Translation: Protein Synthesis

Processing of tRNAs and rRNAs

rRNA and tRNA are structural molecules that aid in protein synthesis but are not themselves translated into protein.

Learning Objectives

Describe how pre-rRNAs and pre-tRNAs are processed into mature rRNAs and tRNAs.

Key Takeaways

Key Points

  • Ribosomal RNA (rRNA) is a structural molecule that makes up over half of the mass of a ribosome and aids in protein synthesis.
  • Transfer RNA (tRNA) recognizes a codon on mRNA and brings the appropriate amino acid to that site.
  • rRNAs are processed from larger pre-rRNAs by trimming the larger rRNAs down and methylating some of the nucleotides.
  • tRNAs are processed from pre-tRNAs by trimming both ends of the pre-tRNA, adding a CCA trinucleotide to the 3′ end, if needed, removing any introns present, and chemically modified 12 nucleotides on average per tRNA.

Key Terms

  • anticodon: a sequence of three nucleotides in transfer RNA that binds to the complementary triplet (codon) in messenger RNA, specifying an amino acid during protein synthesis

Processing of tRNAs and rRNAs

The tRNAs and rRNAs are structural molecules that have roles in protein synthesis; however, these RNAs are not themselves translated. In eukaryotes, pre-rRNAs are transcribed, processed, and assembled into ribosomes in the nucleolus, while pre-tRNAs are transcribed and processed in the nucleus and then released into the cytoplasm where they are linked to free amino acids for protein synthesis.

Ribosomal RNA (rRNA)

The four rRNAs in eukaryotes are first transcribed as two long precursor molecules. One contains just the pre-rRNA that will be processed into the 5S rRNA; the other spans the 28S, 5.8S, and 18S rRNAs. Enzymes then cleave the precursors into subunits corresponding to each rRNA. In bacteria, there are only three rRNAs and all are transcribed in one long precursor molecule that is cleaved into the individual rRNAs. Some of the bases of pre-rRNAs are methylated for added stability. Mature rRNAs make up 50-60% of each ribosome. Some of a ribosome’s RNA molecules are purely structural, whereas others have catalytic or binding activities.

The eukaryotic ribosome is composed of two subunits: a large subunit (60S) and a small subunit (40S). The 60S subunit is composed of the 28S rRNA, 5.8S rRNA, 5S rRNA, and 50 proteins. The 40S subunit is composed of the 18S rRNA and 33 proteins. The bacterial ribosome is composed of two similar subunits, with slightly different components. The bacterial large subunit is called the 50S subunit and is composed of the 23S rRNA, 5S rRNA, and 31 proteins, while the bacterial small subunit is called the 30S subunit and is composed of the 16S rRNA and 21 proteins.

The two subunits join to constitute a functioning ribosome that is capable of creating proteins.

Transfer RNA (tRNA)

Each different tRNA binds to a specific amino acid and transfers it to the ribosome. Mature tRNAs take on a three-dimensional structure through intramolecular basepairing to position the amino acid binding site at one end and the anticodon in an unbasepaired loop of nucleotides at the other end. The anticodon is a three-nucleotide sequence, unique to each different tRNA, that interacts with a messenger RNA (mRNA) codon through complementary base pairing.

There are different tRNAs for the 21 different amino acids. Most amino acids can be carried by more than one tRNA.

.

image

Structure of tRNA: This is a space-filling model of a tRNA molecule that adds the amino acid phenylalanine to a growing polypeptide chain. The anticodon AAG binds the codon UUC on the mRNA. The amino acid phenylalanine is attached to the other end of the tRNA.

In all organisms, tRNAs are transcribed in a pre-tRNA form that requires multiple processing steps before the mature tRNA is ready for use in translation. In bacteria, multiple tRNAs are often transcribed as a single RNA. The first step in their processing is the digestion of the RNA to release individual pre-tRNAs. In archaea and eukaryotes, each pre-tRNA is transcribed as a separate transcript.

The processing to convert the pre-tRNA to a mature tRNA involves five steps.

1. The 5′ end of the pre-tRNA, called the 5′ leader sequence, is cleaved off.

2. The 3′ end of the pre-tRNA is cleaved off.

3. In all eukaryote pre-tRNAs, but in only some bacterial and archaeal pre-tRNAs, a CCA sequence of nucleotides is added to the 3′ end of the pre-tRNA after the original 3′ end is trimmed off. Some bacteria and archaea pre-tRNAs already have the CCA encoded in their transcript immediately upstream of the 3′ cleavage site, so they don’t need to add one. The CCA at the 3′ end of the mature tRNA will be the site at which the tRNA’s amino acid will be added.

4. Multiple nucleotides in the pre-tRNA are chemically modified, altering their nitorgen bases. On average about 12 nucleotides are modified per tRNA. The most common modifications are the conversion of adenine (A) to pseudouridine (ψ), the conversion of adenine to inosine (I), and the conversion of uridine to dihydrouridine (D). But over 100 other modifications can occur.

5. A significant number of eukaryotic and archaeal pre-tRNAs have introns that have to be spliced out. Introns are rarer in bacterial pre-tRNAs, but do occur occasionally and are spliced out.

After processing, the mature pre-tRNA is ready to have its cognate amino acid attached. The cognate amino acid for a tRNA is the one specified by its anticodon. Attaching this amino acid is called charging the tRNA. In eukaryotes, the mature tRNA is generated in the nucleus, and then exported to the cytoplasm for charging.

image

Processing of a pre-tRNA.: A typical pre-tRNA undergoing processing steps to generate a mature tRNA ready to have its cognate amino acid attached. Nucleotides that are cleaved away are shown in green. Chemically-modified nucleotides are in yellow, as is the CAA trinucleotide that is added to the 3′ end of the pre-tRNA during processing. The anticodon nucleotides are shown in a lighter shade of red.

The Protein Synthesis Machinery

Protein synthesis, or translation of mRNA into protein, occurs with the help of ribosomes, tRNAs, and aminoacyl tRNA synthetases.

Learning Objectives

Explain the role played by ribosomes, tRNA, and aminoacyl tRNA synthetases in protein synthesis

Key Takeaways

Key Points

  • Ribosomes, macromolecular structures composed of rRNA and polypeptide chains, are formed of two subunits (in bacteria and archaea, 30S and 50S; in eukaryotes, 40S and 60S), that bring together mRNA and tRNAs to catalyze protein synthesis.
  • Fully assembled ribosomes have three tRNA binding sites: an A site for incoming aminoacyl-tRNAs, a P site for peptidyl-tRNAs, and an E site where empty tRNAs exit.
  • tRNAs (transfer ribonucleic acids), which serve to deliver the appropriate amino acid to the growing peptide chain, consist of a modified RNA chain with the appropriate amino acid covalently attached.
  • tRNAs have a loop of unbasepaired nucleotides at one end of the molecule that contains three nucleotides that act as the anticodon that basepairs to the mRNA codon.
  • Aminoacyl tRNA synthetases are enzymes that load the individual amino acids onto the tRNAs.

Key Terms

  • ribosome: protein/mRNA complexes found in all cells that are involved in the production of proteins by translating messenger RNA

The Protein Synthesis Machinery

In addition to the mRNA template, many molecules and macromolecules contribute to the process of translation. The composition of each component may vary across species. For instance, ribosomes may consist of different numbers of rRNAs and polypeptides depending on the organism. However, the general structures and functions of the protein synthesis machinery are comparable from bacteria to archaea to human cells. Translation requires the input of an mRNA template, ribosomes, tRNAs, and various enzymatic factors.

Ribosomes

A ribosome is a complex macromolecule composed of structural and catalytic rRNAs, and many distinct polypeptides. In eukaryotes, the synthesis and assembly of rRNAs occurs in the nucleolus.

image

The ribosome in action: Structure and role of ribosomes during translation

Ribosomes exist in the cytoplasm in prokaryotes and in the cytoplasm and on rough endoplasmic reticulum membranes in eukaryotes. Mitochondria and chloroplasts also have their own ribosomes, and these look more similar to prokaryotic ribosomes (and have similar drug sensitivities) than the cytoplasmic ribosomes. Ribosomes dissociate into large and small subunits when they are not synthesizing proteins and reassociate during the initiation of translation.E. coli have a 30S small subunit and a 50S large subunit, for a total of 70S when assembled (recall that Svedberg units are not additive). Mammalian ribosomes have a small 40S subunit and a large 60S subunit, for a total of 80S. The small subunit is responsible for binding the mRNA template, whereas the large subunit sequentially binds tRNAs.

In bacteria, archaea, and eukaryotes, the intact ribosome has three binding sites that accomodate tRNAs: The A site, the P site, and the E site. Incoming aminoacy-tRNAs (a tRNA with an amino acid covalently attached is called an aminoacyl-tRNA) enter the ribosome at the A site. The peptidyl-tRNA carrying the growing polypeptide chain is held in the P site. The E site holds empty tRNAs just before they exit the ribosome.

image

Ribosome structure: The large ribosomal subunit sits atop the small ribosomal subunit and the mRNA is threaded through a groove near the interface of the two subunits. The intact ribosome has three tRNA binding sites: the A site for incoming aminoacyl-tRNAs; the P site for the peptidyl-tRNA carrying the growing polypeptide chain; and the E site where empty tRNAs exit (not shown in this figure but immediately adjacent to the P site.)

Each mRNA molecule is simultaneously translated by many ribosomes, all reading the mRNA from 5′ to 3′ and synthesizing the polypeptide from the N terminus to the C terminus. The complete mRNA/poly-ribosome structure is called a polysome.

tRNAs in eukaryotes

The tRNA molecules are transcribed by RNA polymerase III. Depending on the species, 40 to 60 types of tRNAs exist in the cytoplasm. Specific tRNAs bind to codons on the mRNA template and add the corresponding amino acid to the polypeptide chain. (More accurately, the growing polypeptide chain is added to each new amino acid bound in by a tRNA.)

The transfer RNAs (tRNAs) are structural RNA molecules. In eukaryotes, tRNA mole are transcribed from tRNA genes by RNA polymerase III. Depending on the species, 40 to 60 types of tRNAs exist in the cytoplasm. Serving as adaptors, specific tRNAs bind to sequences on the mRNA template and add the corresponding amino acid to the polypeptide chain. (More accurately, the growing polypeptide chain is added to each new amino acid brought in by a tRNA.) Therefore, tRNAs are the molecules that actually “translate” the language of RNA into the language of proteins.

image

The two-dimensional cloverleaf structure of a typical tRNA.: All tRNAs, regardless of the species they come from or the amino acid they carry, self-basepair to produce a cloverleaf structure of four main stems and three main loops. The amino acid carried by the tRNA is covalently attached to the nucleotide at the 3′ end of the tRNA, known as the tRNA’s acceptor arm. The opposite end of the folded tRNA has the anticodon loop where the tRNA will basepair to the mRNA codon.

Of the 64 possible mRNA codons (triplet combinations of A, U, G, and C) three specify the termination of protein synthesis and 61 specify the addition of amino acids to the polypeptide chain. Of the three termination codons, one (UGA) can also be used to encode the 21st amino acid, selenocysteine, but only if the mRNA contains a specific sequence of nucleotides known as a SECIS sequence. Of the 61 non-termination codons, one codon (AUG) also encodes the initiation of translation.

Each tRNA polynucleotide chain folds up so that some internal sections basepair with other internal sections. If just diagrammed in two dimensions, the regions where basepairing occurs are called stems, and the regions where no basepairs form are called loops, and the entire pattern of stems and loops that forms for a tRNA is called the “cloverleaf” structure. All tRNAs fold into very similar cloverleaf structures of four major stems and three major loops.

If viewed as a three-dimensional structure, all the basepaired regions of the tRNA are helical, and the tRNA folds into a L-shaped structure.

image

The three dimensional shape taken by tRNAs.: If viewed as a three-dimensional structure, all tRNAs are partially helical molecules that are vaguely L-shaped. The anticodon-containing loop is at one end of the molecule (in grey here) and the amino acid acceptor arm is at the other end of the molecule (in yellow here) past the bend of the “L”.

Each tRNA has a sequence of three nucleotides located in a loop at one end of the molecule that can basepair with an mRNA codon. This is called the tRNA’s anticodon. Each different tRNA has a different anticodon. When the tRNA anticodon basepairs with one of the mRNA codons, the tRNA will add an amino acid to a growing polypeptide chain or terminate translation, according to the genetic code. For instance, if the sequence CUA occurred on a mRNA template in the proper reading frame, it would bind a tRNA with an anticodon expressing the complementary sequence, GAU. The tRNA with this anticodon would be linked to the amino acid leucine.

Aminoacyl tRNA Synthetases

The process of pre-tRNA synthesis by RNA polymerase III only creates the RNA portion of the adaptor molecule. The corresponding amino acid must be added later, once the tRNA is processed and exported to the cytoplasm. Through the process of tRNA “charging,” each tRNA molecule is linked to its correct amino acid by a group of enzymes called aminoacyl tRNA synthetases. When an amino acid is covalently linked to a tRNA, the resulting complex is known as an aminoacyl-tRNA. At least one type of aminoacyl tRNA synthetase exists for each of the 21 amino acids; the exact number of aminoacyl tRNA synthetases varies by species. These enzymes first bind and hydrolyze ATP to catalyze the formation of a covalent bond between an amino acid and adenosine monophosphate (AMP); a pyrophosphate molecule is expelled in this reaction. This is called “activating” the amino acid. The same enzyme then catalyzes the attachment of the activated amino acid to the tRNA and the simultaneous release of AMP. After the correct amino acid covalently attached to the tRNA, it is released by the enzyme. The tRNA is said to be charged with its cognate amino acid. (the amino acid specified by its anticodon is a tRNA’s cognate amino acid.)

Prokaryotic Transcription and Translation Are Coupled

Prokaryotic transcription occurs in the cytoplasm alongside translation and can occur simultaneously.

Learning Objectives

Describe the events of prokaryotic transcription initiation

Key Takeaways

Key Points

  • In prokaryotes genetic material is not enclosed in a membrane-enclosed nucleus and has access to ribosomes in the cytoplasm.
  • Transcription is known to be controlled by a variety of regulators in prokaryotes. Many of these transcription factors are homodimers containing helix-turn-helix DNA -binding motifs.
  • Additional transcription regulation comes from transcription factors that can affect the stability of the holoenzyme structure at initiation.

Key Terms

  • transcription: The synthesis of RNA under the direction of DNA.

Overview of Prokaryotic Transcription

Prokaryotic transcription is the process in which messenger RNA transcripts of genetic material in prokaryotes are produced, to be translated for the production of proteins. Prokaryotic transcription occurs in the cytoplasm alongside translation. Prokaryotic transcription and translation can occur simultaneously. This is impossible in eukaryotes, where transcription occurs in a membrane-bound nucleus while translation occurs outside the nucleus in the cytoplasm. In prokaryotes genetic material is not enclosed in a membrane-enclosed nucleus and has access to ribosomes in the cytoplasm.

image

Protein synthesis: An overview of protein synthesis.Within the nucleus of the cell (light blue), genes (DNA, dark blue) are transcribed into RNA. This RNA is then subject to post-transcriptional modification and control, resulting in a mature mRNA (red) that is then transported out of the nucleus and into the cytoplasm (peach), where it undergoes translation into a protein. mRNA is translated by ribosomes (purple) that match the three-base codons of the mRNA to the three-base anti-codons of the appropriate tRNA. Newly synthesized proteins (black) are often further modified, such as by binding to an effector molecule (orange), to become fully active.

Transcription is controlled by a variety of regulators in prokaryotes. Many of these transcription factors are homodimers containing helix-turn-helix DNA-binding motifs.

Steps of Transcription Initiation

The following steps occur, in order, for transcription initiation:

  • RNA polymerase (RNAP) binds to one of several specificity factors, σ, to form a holoenzyme. In this form, it can recognize and bind to specific promoter regions in the DNA. The -35 region and the -10 (“Pribnow box”) region comprise the basic prokaryotic promoter, and |T| stands for the terminator.
  • The DNA on the template strand between the +1 site and the terminator is transcribed into RNA, which is then translated into protein. At this stage, the DNA is double-stranded (“closed”). This holoenzyme/wound-DNA structure is referred to as the closed complex.
  • The DNA is unwound and becomes single-stranded (“open”) in the vicinity of the initiation site (defined as +1). This holoenzyme/unwound-DNA structure is called the open complex.
  • The RNA polymerase transcribes the DNA (the beta subunit initiates the synthesis), but produces about 10 abortive (short, non-productive) transcripts which are unable to leave the RNA polymerase because the exit channel is blocked by the σ-factor.The σ-factor eventually dissociates from the holoenzyme, and elongation proceeds.

Additional Transcription Factors

Promoters can differ in “strength”; that is, how actively they promote transcription of their adjacent DNA sequence. Promoter strength is in many (but not all) cases, a matter of how tightly RNA polymerase and its associated accessory proteins bind to their respective DNA sequences. The more similar the sequences are to a consensus sequence, the stronger the binding is.

Additional transcription regulation comes from transcription factors that can affect the stability of the holoenzyme structure at initiation. Most transcripts originate using adenosine-5′-triphosphate (ATP) and, to a lesser extent, guanosine-5′-triphosphate (GTP) (purine nucleoside triphosphates) at the +1 site. Uridine-5′-triphosphate (UTP) and cytidine-5′-triphosphate (CTP) (pyrimidine nucleoside triphosphates) are disfavoured at the initiation site.

Two termination mechanisms are well known: Intrinsic termination (also called Rho-independent transcription termination) involves terminator sequences within the RNA that signal the RNA polymerase to stop. The terminator sequence is usually a palindromic sequence that forms a stem-loop hairpin structure that leads to the dissociation of the RNAP from the DNA template. Rho-dependent termination uses a termination factor called ρ factor(rho factor) which is a protein to stop RNA synthesis at specific sites. This protein binds at a rho utilisation site on the nascent RNA strand and runs along the mRNA towards the RNAP. A stem loop structure upstream of the terminator region pauses the RNAP, when ρ-factor reaches the RNAP, it causes RNAP to dissociate from the DNA, terminating transcription.

The Incorporation of Nonstandard Amino Acids

Aside from the 22 standard amino acids, there are many other amino acids that are called non-proteinogenic or non-standard.

Learning Objectives

Describe the process and function of posttranslational modification

Key Takeaways

Key Points

  • During protein synthesis, 20 different amino acids can be incorporated to become a protein.
  • Posttranslational modification of amino acids change the chemical nature of an amino acid (e.g., citrullination), or make structural changes (e.g., formation of disulfide bridges).
  • Non-standard amino acids either are not found in proteins (e.g., carnitine, GABA), or are not produced directly and in isolation by standard cellular machinery.

Key Terms

  • Posttranslational modification: the chemical modification of a protein after its translation. It is one of the later steps in protein biosynthesis, and thus gene expression, for many proteins.
  • translation: A process occurring in the ribosome, in which a strand of messenger RNA (mRNA) guides assembly of a sequence of amino acids to make a protein.
  • amino acid: Any organic compound containing both an amino and a carboxylic acid functional group.

Posttranslational modification (PTM) is the chemical modification of a protein after its translation. It is one of the later steps in protein biosynthesis, and thus gene expression, for many proteins. A protein (also called a polypeptide) is a chain of amino acids. During protein synthesis, 20 different amino acids can be incorporated to become a protein. After translation, the posttranslational modification of amino acids extends the range of functions of the protein by attaching it to other biochemical functional groups (such as acetate, phosphate, various lipids, and carbohydrates), changing the chemical nature of an amino acid (e.g., citrullination), or making structural changes (e.g., formation of disulfide bridges).

Also, enzymes may remove amino acids from the amino end of the protein, or cut the peptide chain in the middle. For instance, the peptide hormone insulin is cut twice after disulfide bonds are formed, and a propeptide is removed from the middle of the chain; the resulting protein consists of two polypeptide chains connected by disulfide bonds. Also, most nascent polypeptides start with the amino acid methionine because the “start” codon on mRNA also codes for this amino acid. This amino acid is usually taken off during post-translational modification.

Aside from the 22 standard amino acids, there are many other amino acids that are called non-proteinogenic or non-standard. Those either are not found in proteins (e.g., carnitine, GABA), or are not produced directly and in isolation by standard cellular machinery (e.g., hydroxyproline and selenomethionine).

image

Genetic code: The genetic code diagram showing the amino acid residues as target of modification.

Non-standard amino acids that are found in proteins are formed by post-translational modification, which is modification after translation during protein synthesis. These modifications are often essential for the function or regulation of a protein. For example, the carboxylation of glutamate allows for better binding of calcium cations, and the hydroxylation of proline is critical for maintaining connective tissues.

Another example is the formation of hypusine in the translation initiation factor EIF5A through modification of a lysine residue. Such modifications can also determine the localization of the protein. For instance, the addition of long hydrophobic groups can cause a protein to bind to a phospholipid membrane.

It is important to compare the structures of alanine and beta alanine. In alanine, the side-chain is a methyl group; in beta alanine, the side-chain contains a methylene group connected to an amino group, and the alpha carbon lacks an amino group. The two amino acids, therefore, have the same formulae but different structures.

Some nonstandard amino acids are not found in proteins. Examples include lanthionine, 2-aminoisobutyric acid, dehydroalanine, and the neurotransmitter gamma-aminobutyric acid. Nonstandard amino acids often occur as intermediates in the metabolic pathways for standard amino acids. For example, ornithine and citrulline occur in the urea cycle, which is part of amino acid catabolism. A rare exception to the dominance of α-amino acids in biology is the β-amino acid beta alanine (3-aminopropanoic acid), which is used in plants and microorganisms in the synthesis of pantothenic acid (vitamin B5), a component of coenzyme A.

Unsticking Stuck Ribosomes

Ribosomes can get stuck on mRNAs, cells have ways of unsticking them.

Learning Objectives

Differentiate between nonstop mediated decay and trans-translation as mechanisms of freeing stuck ribosomes

Key Takeaways

Key Points

  • Due to errors in mRNAs and degradation of mRNAs, ribosomes can stall on transcripts, the stalling of ribosomes can lead to problems for cells.
  • Nonstop mediated decay, involves the binding of a stuck ribosome by proteins, these proteins free the ribosome and lead to the destruction of the mRNA.
  • Trans-translation involves tmRNA an RNA with the properties of both a tRNA and mRNA, which basically convinces the stuck ribosome that the tmRNA is the next codon of the stuck mRNA. The ribosome then translate the tmRNA, which frees the ribosome.

Key Terms

  • translation: A process occurring in the ribosome, in which a strand of messenger RNA (mRNA) guides assembly of a sequence of amino acids to make a protein.
  • ribosome: Small organelles found in all cells; involved in the production of proteins by translating messenger RNA.

As mRNAs are transcribed a phenomenon of “stuck” or stalled ribosomes can occur. Stuck mRNA transcripts can arise from many different mechanisms such as premature 3′ adenylation or cryptic polyadenylation signals within the coding region of a gene. This lack of a stop codon results a significant issue for cells. Ribosomes translating the mRNA eventually translate into the 3’poly-A tail region of transcripts and stalls. As a result it cannot eject the mRNA. Ribosomes thus may become sequestered associated with the nonstop mRNA and would not be available to translate other mRNA molecules into proteins.

There are two ways in which cells deal with stuck ribosomes, nonstop mediated decay (NSD) and Trans-translation. Nonstop mediated decay mediates this problem by both freeing the stalled ribosomes and marking the nonstop mRNA for degradation in the cell by nucleases. Nonstop mediated decay consists of destroying the nonstop mRNA. The first pathway proteins bind to the stuck ribosome. This binding allows the ribosome to eject the stuck mRNA molecule – this even frees the ribosome and allows it to translate other transcripts. The proteins which freed the ribosome remain with the mRNA which targets the nonstop mRNA for recognition by RNA degradation pathway. NSD is best understood in eukaryotes but similar processes occur in bacteria.

Trans-translation is a recently discovered pathway in E. coli, although it is not completely understood, it involves Transfer- messenger RNA (abbreviated tmRNA) which is a bacterial RNA molecule with dual tRNA-like and messenger RNA-like properties. It is generally agreed that tmRNA first occupies the empty A site of the stalled ribosome. Subsequently, the ribosome moves from the 3′ end of the truncated messenger RNA onto the tmRNA where it translates the codons of the tmRNA until the tmRNA stop codon is encountered. Depending on the organism, the resulting truncated protein is degraded and the truncated mRNA. Trans-translation is essential in some bacterial species, whereas other bacteria require tmRNA to survive when subjected to stressful growth conditions.

image

Trans-translation: rans-Translation stages A through F. A ribosome with its RNA binding sites, designated E, P, and A, is stuck near the 3′ end of a broken mRNA. The tmRNP binds to the A-site, allowing the ribosome to switch templates from the broken message onto the open reading frame of the tmRNA via the resume codon (blue GCA). Regular translation eventually resumes. Upon reaching the tmRNA stop codon (red UAA), a hybrid protein with a proteolysis tag (green beads) is released.